CN105377889B - Heterodimeric proteins - Google Patents

Abstract

In one aspect, the invention provides a heterodimeric antibody comprising a first monomer comprising a first heavy chain constant domain comprising a first variant Fc domain and a first antigen binding domain; the second monomer comprises a second heavy chain constant domain comprising a second variant Fc domain and a second antigen binding domain. In other aspects, the heterodimeric antibody comprises a first monomer comprising a heavy chain comprising a first Fc domain and a single chain Fv region (scFv) that binds a first antigen, wherein the scFv comprises a charged scFv linker. The heterodimeric antibody further comprises a second monomer comprising a first heavy chain comprising a second Fc domain and a first variable heavy chain, and a first light chain.

Description

Heterodimeric proteins

Priority

This application is part of a continuation-in-part application of international patent application No. PCT/US14/11549 filed on 14 th month 1 2014, U.S. patent application No. 14/155,334 filed on 14 th month 1 2014, 14/205,248 filed on 11 th month 3 2014, and 14/207,489 filed on 12 th month 3 2014. Further, the present application claims priority to U.S. patent application No. 61/818,513 filed on day 5/1 2013, 61/818,344 filed on day 5/1 2013, 61/794,896 filed on day 15/3/2013, 61/818,401 filed on day 5/1 2013, 61/913,879 filed on day 9/12/2013, 61/913,832 filed on day 9/12/2013, 61/938,095 filed on day 10/2/2014 and 61/913,870 filed on day 9/12/2013, all of which are specifically incorporated by reference in their entirety, specifically for all figures and legends disclosed herein, and for the amino acid variants disclosed herein.

Background

Antibody-based therapies have been successfully used to treat a variety of diseases, including cancer and autoimmune/inflammatory disorders. However, there is still a need for improvement in such drugs, particularly with respect to enhancing their clinical efficacy. One approach being explored is to engineer additional and novel antigen binding sites into antibody-based drugs such that a single immunoglobulin molecule binds two different antigens together. This non-native or alternative form of antibody that binds two different antigens is often referred to as a bispecific antibody (bispecifics). Because the considerable diversity of antibody variable regions (Fv) makes it possible to produce Fv's that recognize virtually any molecule, a typical way to produce bispecific antibodies is to introduce new variable regions into the antibody.

Many alternative antibody formats have been developed for bispecific targeting (Chames)&Baty,2009,mAbs 1[6]:1-9；Holliger&Hudson,2005,Nature Biotechnology 23[9]1126 + 1136; kontermann, mAbs 4(2):182(2012), all of which are specifically incorporated herein by reference). Initially, bispecific antibodies were prepared by fusing two cell lines, each of which produced a single monoclonal antibody (Milstein et al, 1983, Nature 305: 537-540). Although the resulting hybrid hybridomas or quadromas do produce bispecific antibodies, they are only a small population and require extensive purification to isolate the desired antibody. An engineering solution to this is to make bispecific antibodies using antibody fragments. Because such fragments lack the complex quaternary structure of the full-length antibody, the light and heavy chain variable regions can be joined in a single genetic construct. Many different forms of antibody fragments have been generated, including diabodies, single chain antibodies, tandem scFv's, and Fab₂Dual specificitiesSex antibodies (Chames)&Baty,2009,mAbs 1[6]:1-9；Holliger&Hudson,2005,Nature Biotechnology 23[9]1126 + 1136; specifically incorporated herein by reference). Although these forms can be expressed at high levels in bacteria and can have beneficial osmotic benefits due to their small size, they are rapidly cleared in vivo and can present manufacturing obstacles related to their production and stability. The main reason for these disadvantages is that antibody fragments typically lack the constant regions of the antibody and their associated functional properties, including larger size, high stability, and binding to various Fc receptors and ligands that maintain long half-lives in serum (i.e., the neonatal Fc receptor FcRn) or serve as binding sites for purification (i.e., protein a and protein G).

More recent work has attempted to address the drawbacks of fragment-based bispecific antibodies by modifying dual binding to full-length antibody-like forms (Wu et al, 2007, Nature Biotechnology 25[11]: 1290-1297; USSN12/477,711; Michaelson et al, 2009, mAbs 1[2]: 128-141; PCT/US 2008/074693; Zuo et al, 2000, Protein Engineering 13[5]:361 367; USSN09/865,198; Shen et al, 2006, J Biol Chem 281[16]: 10706-10714; L u et al, 2005, J Biol Chem 280[20]: 19665-19672; PCT/US 2005/025472; specifically incorporated herein by reference.) these forms overcome some of the obstacles of antibody fragment bispecific antibodies, primarily because they contain a Fc region and thus a constant binding site for these antigen dimeric forms are always a top of the constant binding of the new antigenic dimers.

For many antigens attractive as common targets in the form of therapeutic bispecific antibodies the required binding is monovalent rather than bivalent for many immune receptors the cell activation is achieved by cross-linking of monovalent binding interactions the mechanism of cross-linking is typically mediated by antibody/antigen immune complexes or by engagement of effector cells with target cells for example low affinity Fc γ receptors (Fc γ Rs) such as Fc γ RIIa, Fc γ RIIb and Fc γ RIIIa bind to the antibody Fc region monovalent binding does not activate cells expressing these Fc γ Rs, however in immuno-complexation or cell-cell contact the receptors are cross-linked and aggregated on the cell surface resulting in activation for receptors responsible for mediating cell killing, e.g. Fc γ -reservoir on Natural Killer (NK) cells, receptor cross-linking and cell activation occurs when effector cells engage target cells in high affinity (avid) form, and receptor cross-linking occurs when effector cells engage target cells in high affinity (Bowles & Weiner,2005, J-mull 304:88-99, particularly by citation of the same on the cited cell surface, J-receptor, 8: 88-99, when effector cells engage target cells in high affinity (wt) with target cells only the same as well as the antigen receptor binding of effector cells 35, the antigen receptor, it is specifically incorporated in the clinically by the mechanism of effector cells 35 receptor for activation of effector cells, (ii) the antigen receptor binding to stem cell-effector cells, (c-receptor binding to stem-effector cells) the antigen receptor, (ii receptor, the antigen receptor, the stem-receptor binding of stem-effector cells, (c receptor, the stem-effector cells) is preferably for the stem-effector cells, (ii receptor, the stem-effector cells, (ii) is specifically binds to activate the stem-effector cells, (ii) when the stem-effector cells, (c-cell-effector cells, (ii) is specifically, the stem-cell binding to activate the stem-effector cells, (c-cell-effector cells, (ii) the stem-effector cells, (ii) is) to activate the stem-effector cells, (c receptor, (ii) the stem-cell-effector cells, (c) the stem-effector cells, (c receptor, (c) the stem-cell binding of stem-cell binding to activate the stem-effector cells, (c receptor, (c) the stem-effector cells, (c) the stem-cell binding of stem-stem cell binding of stem-stem cell binding of stem-stem cell binding of stem-stem.

Thus, while bispecific antibodies generated from antibody fragments encounter biophysical and pharmacokinetic barriers, those constructs having full-length antibody-like formats suffer from the disadvantage that in the absence of the primary target antigen, they multivalently engage a common target antigen, resulting in nonspecific activation and potential toxicity. The present invention solves this problem by introducing a new set of bispecific antibody formats that are capable of multivalent co-conjugation to different target antigens. In addition, the present invention provides novel heterodimeric variants that allow for better formation and purification of heterodimeric proteins, including antibodies.

Brief description of the invention

In one aspect, the invention provides a heterodimeric antibody comprising a first monomer comprising a first heavy chain constant domain comprising a first variant Fc domain and a first antigen binding domain; the second monomer comprises a second heavy chain constant domain comprising a second variant Fc domain and a second antigen binding domain.

In other aspects, the heterodimeric antibody comprises a first monomer comprising a heavy chain comprising a first Fc domain and a single chain Fv region (scFv) that binds a first antigen, wherein the scFv comprises a charged scFv linker. The heterodimeric antibody further comprises a second monomer comprising a first heavy chain comprising a second Fc domain and a first variable heavy chain, and a first light chain. In other aspects, the charged linker bears a positive charge of 3-8 or a negative charge of 3-8 and is selected from the group consisting of those linkers depicted in fig. 9.

In a further aspect, the invention provides a heterodimeric antibody composition comprising a first monomer comprising a first heavy chain sequence comprising a first variant Fc domain as compared to a human Fc domain; and a first antigen binding domain that binds a first antigen; the second duplex sequence comprises: a second variant Fc domain compared to a human Fc domain; and a second antigen-binding domain that binds a second antigen; wherein the first and second variant Fc domains comprise a set of amino acid substitutions selected from the group consisting of the amino acid sets depicted in FIG. 3.

In other aspects, the present invention provides heterodimeric antibody compositions comprising a first monomer comprising a first heavy chain sequence comprising a first variant Fc domain compared to a human Fc domain and a second heavy chain sequence comprising a second variant Fc domain compared to a human Fc domain and a second antigen-binding domain that binds CD19, the second antigen-binding domain comprising a variable heavy chain domain comprising an amino acid sequence of H1.227 (SEQ ID NO: X) and a variable light chain selected from the group consisting of an amino acid sequence of L1.198 (SEQ ID NO: X) and an amino acid sequence of 1.199 (SEQ ID NO: X) as depicted in FIG. 21.

In A further aspect, the invention provides A heterodimeric antibody composition comprising A first monomer comprising A first heavy chain sequence comprising A first variant Fc domain compared to A human Fc domain and A first antigen-binding domain comprising an anti-CD variable region having A sequence comprising A vhCDR, A vCDR and A vCDR, wherein the vhCDR has the sequence T-Y-A-M-XaA, wherein XaA is or H (SEQ ID NO:435), A light chain variable domain comprising the sequence R-I-R-K-XaA-N-XaA-Y-A-T-XaA-Y-Y-A-XaA-S-V-K-G, wherein XaA is Y or A, XaA is N or S, XaA is Y or A, XaA is or A, and XaA is or A (XaA is or A) and XaA is or A, and A is or A, and the light chain variable domain comprises the sequence of the anti-CD variable Fc domain of the sequence of the anti-CD variable region comprises the anti-CD variable region of the sequence of the vhCDR-CD (vhCDR-CD) and the sequence of the vH-CD variable region comprising the vCDR, wherein XaA-CD variable region of the sequence of the vhCDR-CD (vCDR, the sequence of the vCDR-CD (vCDR, the sequence of the vCDR, wherein XaA-CD variable region of the sequence of the vCDA-CD-H-CD (SEQ ID-H-CD) has the sequence of the vCDR, the sequence of the vCDA-H, the sequence of the light chain (SEQ ID, the light chain (SEQ ID NO: SEQ ID of the sequence of the vCDA-H-A-H-A-H, the light chain (SEQ ID NO: the vCDR, the light chain of the sequence of the light chain of the vH-H-X (SEQ ID NO: the vH-H-X, the light chain (SEQ ID NO: 1-X, the light chain of the amino acid of the vH-X, the light chain of the vH-H-X-H-X, the sequence of the light chain of the amino acid (SEQ ID NO: the vH-X-H-X, the amino acid (SEQ ID NO: the amino acid-X, the light chain of the amino acid-X, the sequence of the amino acid sequence of the vH-X, the amino acid sequence of the amino acid of the light chain of the amino acid of the vH-H-X (SEQ ID NO: the amino acid of the amino acid sequence of the amino acid of.

In other aspects, the invention provides heterodimeric antibodies comprising a first monomer comprising a heavy chain comprising a first variant Fc domain; and a single chain Fv region (scFv) that binds a first antigen, wherein said scFv comprises a charged scFv linker; the second monomer comprises a first heavy chain comprising a second variant Fc domain and a first variable heavy chain, and the second monomer further comprises a first light chain, wherein the first and second variant Fc domains comprise an amino acid substitution(s) selected from the group consisting of those depicted in figure 7.

In A further aspect, the invention provides an heterodimeric antibody composition comprising A first monomer comprising A first antigen-binding domain comprising an anti-CD variable region comprising A vhCDR, vCDR and vCDR, wherein the XaA is the hCDR-vA or the vCDR-vA domain, wherein the XaA is the H-CDR or the vS-CDR domain, or the XaA is the H-T-A-M-XaA is the H (SEQ ID NO:435) or the vA-CD variant wherein the XaA is the H-T-S or the XaA vA-vS-H-T-A-T-XaA-Y-Y-A-Y-A-S-S-V-K-G, wherein the XaA is the XaA Y or A is the vA or the XaA-T-A-V-XaA variant, wherein the XaA is the vT-I or the vR-I-A-I-V-I (or the vA-I, or the XaA-I, or the XaA-I, wherein the XaA-I (or the XaA-I, or the XaA-I, wherein the XaA-I, or the XaA-I, or the XaA-I, wherein the XaA is the XaA-I, or the XaA-I, or the vA-I, or the XaA-I, or the vA-I, the XaA-I, or the XaA-I, the vA-I, the XaA-I, the vA, or the XaA-I, or the XaA, the XaA-I, or the XaA-I, or the XaA-I, or the XaA (or the XaA, the vA, the XaA, or the XaA, the XaA-I, the XaA, or the vA, or the XaA, the XaA (or the vA, the XaA, the vA, the XaA, the vA, the vS-A, the vA, the vS-I, the XaA, the vS-A, the vS-I, the vS-A, the vS, the vA, the XaA, the vS, the vA, the vS, the (SEQ ID, the vS, the XaA, the (or the vA, the vS-A, the (SEQ ID, the vA, the (or the vS, the XaA, the (SEQ ID, the XaA, the.

In a further aspect, the present invention provides a heterodimeric protein comprising a first monomer comprising a first variant heavy chain constant region and a first fusion partner, and a second monomer comprising a second variant heavy chain constant region and a second fusion partner, wherein the Fc regions of the first and second constant regions comprise a set of amino acid substitutions from FIGS. 3 and 12.

In many aspects, one of the first and second variant Fc domains comprises the amino acid substitution (S) selected from the group consisting of those depicted in fig. 6,7, and/or 12 in some aspects the first antigen binding domain is an scfv covalently attached to the first heavy chain constant domain in other aspects the heterodimeric antibody has a structure selected from the structures of fig. 1B-1L and 2A-2M in yet further aspects the first and/or second Fc domains of the heterodimeric antibody further comprises the amino acid substitution (S) selected from the group consisting of 434A,434S, 428L, 308F,259I,428 2/434S, 259I/308F, 436I/428/L, 436I or V/434S,436V/428 5, 252Y/254T/256E, 259I/L, 308A, 239D,239E, 239D, 267F, 267I/320, 267I/76F, 267I/or V/243S, 267F, 267F, 267F, 267F, 267.

In other aspects, the invention provides nucleic acids, expression vectors and host cells that will produce the heterodimeric proteins and antibodies of the invention.

In a further aspect, the invention provides a method of making a heterodimeric protein of the invention by culturing a host cell comprising a nucleic acid encoding the heterodimeric protein of the invention and an antibody under conditions wherein the heterodimer is produced and recovering the heterodimer.

In a further aspect, the invention provides a method of making a heterodimeric antibody of the invention, the method comprising: providing a first nucleic acid encoding a first heavy chain comprising: a first Fc domain; and a single chain Fv region (scFv) that binds a first antigen; wherein the scFv comprises a charged linker; and providing a second nucleic acid encoding a second heavy chain comprising: a second Fc domain; a first variable heavy chain; and providing a third nucleic acid comprising a light chain. The method further comprises expressing the first, second and third nucleic acids in a host cell to produce first, second and third amino acid sequences, respectively; loading the first, second, and third amino acid sequences onto an ion exchange column; and collecting the heterodimer fraction.

In other aspects, the invention provides methods of treating an individual in need of such treatment by administering a heterodimeric antibody or protein herein.

Brief Description of Drawings

Heterodimeric forms and variants

FIGS. 1A-1M depict a number of heterodimeric protein forms, including heterodimeric Fc fusion proteins and heterodimeric antibodies FIG. 1A shows the basic concept of a dimeric Fc region with 4 possible fusion partners A, B, C and D are optionally and independently selected from immunoglobulin domains (e.g., Fab, vH, v L, scFv)₂scFab, dAb, etc.), peptides, cytokines (e.g., I L-2, I L-10, I L-12, GCSF, GM-CSF, etc.), chemokines (e.g., RANTES, CXC L, CXC L, CXC L, etc.), hormones (e.g., FSH, growth hormone), immunoreceptors (e.g., CT L a-4, TNFR1, TNFRII, other TNFSF, other TNFRSF, etc.) and blood factors (e.g., factor VII, factor VIII, factor IX, etc.) as outlined herein, domains filled with solid white or solid black are engineered with heterodimeric variants, figure 1B depicts an "F triplet" format (also sometimes referred to as a "F triplet" format, as discussed below) figure 1C shows an "F triplet" format with another scFv linked to a Fab monomer (this one, with figure 1F, also having a larger triplet "format, as depicted in figure 1D) that has a single scFv linkage to a scFv 1H-scFv-1H" scFv-1 antigen binding scheme that is depicted as a double scFv-1H-scFv-1H-1H-scFv-1, and figures that depicts a double scFv-1H-G-1-G.

FIG. 2A-2U depicts a number of multispecific (e.g. heterodimeric) forms and combinations of different types of heterodimeric variants that may be used in the present invention (these are sometimes referred to herein as "heterodimeric scaffolds"), further note that all of these forms may include additional variants in the Fc region, as discussed more fully below, including "eliminated" or "knocked out" variants (FIG. 7), Fc variants that alter Fc γ R (Fc γ RIIb, Fc γ RIIIa, etc.) binding, Fc variants that alter binding to FcRn receptor, etc. FIG. 2A shows a double scFv-Fc form, for all heterodimeric forms herein, which may include heterodimeric variants such as pI variants, knob in well (KIH, also referred to herein as hindered or "skewed" variants "), charge pairs (variants of hindered variants), alternative heavy chain variants, and SEED bodies (" chain exchange engineered domains "; see Klein et al, mAb 4: 53-pro-scFv) and heavy chain fusion Protein, heavy chain fusion, heavy chain, heavy.

Figure 3 depicts a number of suitable heterodimerization variants for use in the heterodimeric proteins of the present invention, including skew/steric hindrance variants, isosteric variants, pI variants, KIH variants, and the like. For all heterodimeric structures herein, each set of these heterodimeric variants can be combined in any heterodimeric backbone, optionally and independently in any combination. The variants at the end of the monomer 1 list are isosteric pI variants, which are not normally used in pairs or groups. In this case, one monomer is engineered to increase or decrease the pI without changing the other monomer. Thus, although depicted in the list of "monomer 1", these may be incorporated into suitable monomers, leaving the "chain" type. That is, while the variants listed as "monomer 1" variants in the steric list can cross over the "monomer 2" variants in the pI list, it is important that the "strands" of the monomer pairs remain intact. That is, any group may be combined with any other group, regardless of their associated list of "monomers" (as discussed more fully below, in the case where a change in pI is used to purify the heterodimeric protein, the "pI chain" is also retained; for example, in the case of a liquid, if there are skewed variants that happen to change charge, they pair with pI variants on the correct strand; the bias variant that results in an increase in pI is added to the monomer to which the pI variant has been added, etc. this is similar to the addition of a charged scFv linker; in the case of this situation, it is, in addition, each pair of amino acid variants (or where there is a single monomer engineered therein) may optionally and independently be included or excluded by any heterodimeric protein, and may optionally and independently be combined.

Fig. 4A,4B and 4C depict the heterodimerization variant subgroup of fig. 3 that is particularly useful in the present invention.

Fig. 5 depicts a subset of the heterodimerized variants of fig. 3.

Figure 6 depicts a list of isotype and isosteric variant antibody constant regions and their respective substitutions. pI _ (-) indicates the lower pI variant, and pI _ (+) indicates the higher pI variant. These may optionally and independently be combined with other heterodimerization variants of the invention.

For many, if not all, variants herein, these KO variants can independently and optionally be within the groups described in figure 35, and combined with any of the heterodimerization variants outlined herein (including steric and pI variants). for example, E233P/L V/L a/G236del can be combined with any other single or two variants from the list.

Figure 8 depicts a number of anti-CD 3 scFv engineered disulfides.

Figure 9 depicts a number of charged scFv linkers for increasing or decreasing the pI of heterodimeric proteins that utilize one or more scfvs as components. A single prior art scFv linker with a single charge is "Whitlow" from Whitlow et al Protein Engineering 6(8): 989. sup. 995 (1993). It should be noted that this linker serves to reduce aggregation of the scFv and enhance proteolytic stability.

Fig. 10A and 10B are additional lists of potential heterodimerization variants for use in the invention, including isotype variants.

FIG. 11 depicts a matrix of possible combinations of heterodimeric forms, heterodimeric variants (divided into pI variants and hindered variants (which include charge pair variants)), Fc variants, FcRn variants, and combinations) legend A is a suitable FcRn variant: 434A,434S, 428L, 308F,259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y,252Y/254T/256E and 259I/308F/L. that the FcRn triplet form of FIG. 1B may have either of these FcRn variants on either or both monomer sequences FcRn variants for clarity, FcRn variants (and Fc variants) may be present on one or both monomers as FcRn variants due to the fact that the respective heavy chains are different FcRn variants are for clarity, 267D variants) are suitable Fc variants 236A,239D,239E,332E, 332D/267E, 267D, 328F variants, 328, or 76F/328, 236F/320F variants, and the legend for the possible combinations of hindered variants are read in the concise or for the deletion of the Fc variants and the deletion of the Fc variants and deletion of the Fc variants in the deletion of the deletion.

FIGS. 12A-12J depict additional heterodimerization variant pairs.

Specific sequences of the invention

FIG. 13 depicts the amino acid sequences of the wild-type constant region and IgG1/G2 fusions used in the present invention.

FIGS. 14A-14YY depict the amino acid sequences, variable heavy and variable light chain sequences of a humanized anti-CD 3 variant scFv with optimized stability. (Note also that the first sequence is a histidine-tagged version for ease of purification). CDRs are underlined. It is understood that the increased stability of the optimized variable and optimized light chains (as well as scFv chains) can be attributed to the framework regions as well as the CDRs. Thus, it should be understood that disclosure of the entire variable region includes disclosure of framework regions, although they are not numbered individually. Additionally, scFv linkers are shown in grey. Each scFv linker can be replaced with a charged scFv linker, as depicted in figure 5. That is, any charged scFv linker, whether positively or negatively charged, including those depicted in fig. 5, can replace the highlight region in fig. 3A-3 YY.

FIGS. 15A-15I depict the ordering rules for all CD3 vhCDR1-3 and vlCDR1-3 sequences useful in the present invention. The sequence of the consensus CDR is shown at the end of the figure. FIG. 6 depicts

Figure 16 shows the sequence of XENP13790, which is XENP12912(CD3 scFv + disulfide) with a charged linker added.

FIGS. 17A,17B and 17C. Fig. 17A depicts two different F triplet embodiments. Fig. 17B and 17C show the sequence of the F triplet embodiment of fig. 17A.

FIG. 18 depicts the sequences of a preferred embodiment of the present invention. The variable regions are underlined and the charged scFv linker is grey.

Fig. 19A and 19B. Stability optimized humanized anti-CD 19 variant scFv Tm and Tm changes. The amino acid numbering convention is Kabat numbering system. FIG. 19A is an assay for the stability of humanized anti-CD 19 variant scFv by DSF (differential scanning fluorescence) at a concentration of 0.2mg/ml and FIG. 19B at 0.4 mg/ml.

FIGS. 20A-20K. Amino acid sequences of stability-optimized humanized anti-CD 19 variant scFv, variable heavy chain and variable light chain sequences. (Note also that the first sequence is a histidine-tagged version for ease of purification). It is understood that the increased stability of the optimized variable and optimized light chains (as well as scFv chains) can be attributed to the framework regions as well as the CDRs. Thus, it should be understood that disclosure of the entire variable region includes disclosure of framework regions, although they are not numbered individually.

FIG. 21 depicts the stabilized anti-CD 19 Fv region.

FIGS. 22A and 22B depict a bis-scFv construct (e.g., as shown in FIG. 1M).

Fig. 23A and 23B depict a "bottle opener" construct (e.g., as shown in fig. 1B).

FIGS. 24A-24K show additional sequences of the invention including isosteric dimeric variants.

Data information

FIG. 25. stabilized anti-CD 19 variable domains-competed for binding with labeled anti-CD 19 IgG1@ 1. mu.g/m L.

FIG. 26 shows the characterization and comparison of the bis scFv-Fc format, anti-CD 3/anti-CD 19 versus the "BiTE" format (using the same scFv but without the Fc region). As shown, bis scFv-Fc is not as effective as the BiTE format, but the addition of the Fc region increases half-life 10-fold in mice.

Figure 27 depicts scFv moieties each cross-reacting with cynomolgus monkey antigens in RTCC assays. That is, differences in efficacy between the various forms (bis-scFv-Fc vs. BiTE) were translated into cynomolgus monkeys.

Figure 28 shows that the half-life difference was also transformed into cynomolgus monkeys, as between the two forms. As shown, the bis scFv-Fc antibody was tested at three different concentrations.

Figure 29 depicts prediction of pharmacokinetics in monkeys for longer durations than BiTE at serum concentrations greater than EC50 for the bis scFv-Fc form, 2-3 weeks compared to 2-3 days.

FIG. 30 shows the greater and prolonged B cell killing using the bis scFv-Fc bispecific format. Longer PK in this form is able to prolong B cell depletion to 14 days.

FIG. 31 depicts the stability engineering of the anti-CD 3/anti-CD 19 scFv-Fc scFv portion A substantial improvement in stability was achieved by the recognition and substitution of rare amino acids, the recognition and substitution of amino acids with unusual contact residues, and the engineering and conversion of the linker into the V L-VH orientation.

Figure 32 depicts a doubling of half-life in mice due to PK enhancement induced in mice by stabilization for anti-CD 19 stabilization.

FIG. 33 shows the preparation and purification of "F triad" or "bottle opener" (or Fab-scFv-Fc in some figures).

Figure 34 shows a characterization of the anti-CD 19/anti-CD 3F triplet form, which exhibits picomolar cytotoxicity, binding only monovalently to the target antigen.

FIG. 35 shows the PK increase in mice caused by replacing one scFv of the bis scFv-Fc with Fab replacement of the anti-CD 19 scFv with Fab doubles the half-life in B L/6 mice from 3 days to 6 days.

FIG. 36 depicts a scheme of a "plug and play platform" in the form of F triplets. Fab from any of the existing mAbs can be combined with an anti-CD 3 scFv-Fc bispecific format.

Figures 37A and 37B depict the characterization of a "plug and play" combination of an existing antibody in combination with the F triplet form. FIG. 41A shows the F triplet form of anti-CD 38 Fab and anti-CD 3 scFv and FIG. 41B shows the Her2/CD3 combination.

Figure 38 depicts that using the variants of the present invention significantly "skewed" towards heterodimerization, greater than 95% heterodimerization was achieved using one monomer with L368E/K370T and another monomer with S364K compared to the same molecule without the Fc variant.

Figure 39 shows B cell deficiency in cynomolgus monkey lymph nodes and spleen using bis scFv format compared to BiTE format.

FIG. 40 list of bevacizumab, Fc only and anti-CD 19xCD3 heterodimers containing isosteric pI substitutions. The pI values for each desired protein species are indicated.

Figure 41 cation exchange chromatography showing purification of heterodimeric species of bevacizumab containing isosteric engineered constant regions.

Figure 42 cation exchange chromatography showing purification of heterodimeric species of Fc-only variants containing isosteric engineered constant regions.

Figure 43 cation exchange chromatography showing purification of heterodimeric species of an anti-CD 19xCD3 bispecific antibody containing isosteric engineered constant regions. Also shown are protein a purified material and IEF gels of isolated heterodimeric bispecific antibodies.

Figure 44 list of bevacizumab and Fc-only variants containing isosteric pI substitutions and Tm values obtained from DSF.

FIG. 45 List of anti-CD 3 and anti-CD 19 scFvs containing a positively charged linker and a negatively charged linker. DSF Tm values are also shown.

Figure 46 example SEC chromatograms from purified scFvs having a linker with a positive charge.

FIG. 47 direct binding of anti-CD 3 scFv containing a positively charged linker to CD4+ T cells (left) or CD20+ cells from PBMCs (to check for non-specific binding; right).

FIG. 48 direct binding of anti-CD 3 scFv containing a positively charged linker to CD20+ cells (left) or 293E cells (right) from PBMCs.

Figure 49 example cation exchange purification of XENP13124, XENP13124 is a Fab-scFv-Fc format bispecific antibody targeting CD19 and CD 3. The anti-CD 3 scFv contained a positively charged linker (GKPGS)4 to enable purification.

Figure 50 example SEC chromatograms of purified Fab-scFv-Fc format bispecific antibodies targeting CD19 and CD3 incubated at different concentrations. XENP13121 (left) contains a standard (GGGGS)4 linker and XENP13124 (right) contains a (GKPGS)4 charged linker. The charged linker has the unexpected property of reducing the amount of polymer aggregates present.

FIG. 51 RTCC assay using PBMC and a Fab-scFv-Fc format bispecific anti-CD 19xCD3 antibody with different scFv linkers. The linker had little effect on RTCC activity, except for the highly charged linker (gkgkgkks) 3, which had lower activity.

FIGS. 52A-52O show sequences of the invention comprising charged scFv linkers and corresponding controls.

Miscellaneous other materials

FIG. 53.20 literature pI of amino acids. It should be noted that the listed pI's are calculated as free amino acids; the actual pI of any side chain is different in the case of proteins, and therefore this list is used to show pI trends and is not an absolute number for the purposes of the present invention.

Fig. 54A, 54B and 54c list of all possible pI-reduced variants formed by isotype substitutions of IgG 1-4. pI values for the three desired species are shown, as well as the average Δ pI between the heterodimer and the two homodimer species present when the variable heavy chain was transfected with IgG1-WT heavy chain.

FIG. 55 list of all possible increased pI variants formed by isotype substitutions of IgG 1-4. pI values for the three desired species are shown, as well as the average Δ pI between the heterodimer and the two homodimer species present when the variable heavy chain was transfected with IgG1-WT heavy chain.

Figure 56 shows the amino acid sequences of CK and C λ light chain constant regions. Residues contributing to higher pI (K, R and H) or lower pI (D and E) are highlighted in bold. Preferred positions for modifications to reduce pI are shown in grey. For backbones containing one or more light chains, these changes can be used to alter the pI of one monomer or both monomers, and can be combined independently and optionally with all heavy chain variants.

FIGS. 57A-57E depict the sequences of a number of disulfide constructs; the first sequence was an scFv construct comprising a His (6) tag that facilitated purification, the second sequence was an scFv construct without a tag, the third sequence was an individual variable heavy chain, and the fourth sequence was an individual variable light chain sequence. CDRs are underlined.

Detailed Description

FIGS. 66-80, the sequence and the accompanying legend of WO/2013/055809 are specifically incorporated herein by reference. Fig. 2-111 from USSN 13/648,951 and their legend are specifically incorporated herein by reference.

I. Review of heterodimerization proteins

The present invention relates to novel constructs that provide heterodimeric proteins that allow binding to more than one antigen or ligand, e.g., to allow multi-specific binding. The heterodimeric protein constructs are based on the self-assembly properties of the two Fc domains of the heavy chain of an antibody, e.g., two "monomers" that assemble into a "dimer". Heterodimeric proteins are prepared by altering the amino acid sequence of each monomer, as discussed more fully below. Thus, the present invention is generally directed to the formation of heterodimeric proteins (including antibodies) that can co-engage an antigen in several ways, depending on the amino acid variants in the constant region that differ on each chain to promote heterodimer formation and/or to allow easier purification of heterodimers relative to homodimers. As discussed more fully below, the heterodimeric protein may be an antibody variant or an Fc-based fusion protein. Typically, heterodimeric antibodies are the focus of discussion, but as will be understood by those skilled in the art and described more fully below, the discussion applies equally to heterodimeric proteins.

Accordingly, the present invention provides bispecific antibodies (or, as discussed below, trispecific or tetraspecific antibodies can also be prepared). A continuing problem in antibody technology is the need for "bispecific" (and/or multispecific) antibodies that bind simultaneously to two (or more) different antigens, typically thereby allowing the different antigens to access and generate new functionalities and new therapies. Typically, these antibodies are prepared by including the genes for each of the heavy and light chains into a host cell. This generally results in the formation of the desired heterodimer (A-B), as well as two homodimers (A-A and B-B). However, a major obstacle to the formation of multispecific antibodies is the difficulty in purifying heterodimeric antibodies from homodimeric antibodies and/or the bias towards heterodimer formation in homodimeric formation.

There are many mechanisms that can be used to generate the heterodimers of the present invention. In addition, as will be appreciated by those skilled in the art, these mechanisms can be combined to ensure a high degree of heterodimerization. Thus, amino acid variants that result in the production of heterodimers are referred to as "heterodimerization variants". As discussed below, heterodimerization variants may include steric variants (e.g., "knob and hole" or "skew" variants described below and "charge pair" variants described below) as well as "pI variants" which allow separation of homodimer from heterodimer purification.

One mechanism, often referred to in the art as "knob and hole" ("KIH"), or sometimes referred to herein as a "skew" variant, refers to amino acid engineering that creates steric effects to favor heterodimer formation and may also optionally use amino acid engineering that does not favor homodimer formation; this is sometimes referred to as "knobs and holes" as in USSN 61/596,846 and USSN 12/875,0015, Ridgway et al, Protein Engineering 9(7):617 (1996); atwell et al, J.mol.biol.1997270: 26; US patent no 8,216,805, US 2012/0149876, all of which are incorporated herein by reference in their entirety. The figure identifies a number of "monomer a-monomer B" pairs, which include "knob and hole" amino acid substitutions. In addition, these "knob and hole" mutations can be combined with disulfide bonds to bias formation towards heterodimerization as described in Merchant et al, Nature Biotech.16:677 (1998).

One other mechanism useful in the generation of heterodimers is sometimes referred to as "electrostatic steering" or "charge pairing" as described in Gunasekaran et al, j.biol. chem.285(25):19637(2010), herein incorporated by reference in its entirety, which is sometimes referred to herein as "charge pairing". in this embodiment, formation is biased towards heterodimerization using static electricity, as will be understood by those skilled in the art, these may also have an effect on pI and thus purification, and thus may also be considered pI variants in some cases, however, since these are generated for forced heterodimerization and are not used as purification tools, they are classified as "steric variants". these include, but are not limited to, the pairing of D221E/P E/L E with D221R/P R/K409 (e.g., these are "monomer correspondences") and the pairing of C221/P228 with C483228/4832/K409/224 and C63220/224K 63220/224.

In the present invention, in some embodiments, pI variants are used to alter the pI of one monomer or two monomers and thus allow isoelectric purification of a-A, A-B and B-B dimer proteins.

In the present invention, there are several basic mechanisms that can lead to easy purification of heterodimeric proteins; one relies on the use of pI variants such that each monomer has a different pI, thereby allowing isoelectric purification of a-A, A-B and B-B dimeric proteins. Alternatively, some backbone forms, such as the "F triplet" form, also allow for size-based separation. As outlined further below, heterodimer formation can also be skewed over homodimer. Thus, combinations of sterically hindered heterodimerization variants and pI or charge pair variants are particularly suitable for use in the present invention. In addition, as outlined more fully below, utilizing a backbone of the scFv, such as the F triplet format, can include a charged scFv linker (positively or negatively charged), which provides further pI elevation for purification purposes. As will be appreciated by those skilled in the art, some of the F triplet forms are useful with just a charged scFv linker and do not require additional pI adjustments, although the invention does also provide for the use of skewed variants (as well as combinations of Fc, FcRn and KO variants) with charged scFv linkers.

In the present invention that utilizes pI as a separation mechanism to allow purification of heterodimeric proteins, amino acid variants can be introduced into one or both monomeric polypeptides; that is, the pI of one monomer (referred to herein for simplicity as "monomer a") can be engineered to be much different from that of monomer B, or both monomers a and B can be altered, with the pI of monomer a increasing and the pI of monomer B decreasing. As described in more detail below, the pI changes for either or both monomers can be accomplished as follows: removal or addition of charged residues (e.g., natural amino acids are replaced with positively or negatively charged amino acid residues, e.g., glycine to glutamic acid), changing charged residues from positively or negatively charged to the opposite charge (aspartic acid to lysine) or changing charged residues to neutral residues (e.g., lost charge; lysine to serine). Many such variations are shown in the drawings.

Thus, in this embodiment of the invention it is provided that sufficient pI change is formed in at least one monomer such that heterodimers can be separated from homodimers. As will be appreciated by those skilled in the art, and as discussed further below, this may be accomplished by: "wild-type" heavy chain constant and variable regions that have been engineered to increase or decrease their pI (wt A- + B or wt A-B), or to increase one region and decrease the other (A + -B-or A-B +), are used.

Thus, in general, a component of some embodiments of the invention is an amino acid variant in the constant region of an antibody that involves altering the isoelectric point (pI) of at least one, if not both, of the monomers of the dimeric protein to form "pI heterodimers" (when the protein is an antibody, these are referred to as "pI antibodies") by adding amino acid substitutions ("pI variants" or "pI substitutions") to one or both of the monomers. As shown herein, separation of heterodimers from homodimers can be achieved if the pI of the two monomers differ by as little as 0.1 pH units, and in the present invention the pI of the two monomers differ by more than 0.2, 0.3, 0.4, and 0.5 can all be used.

As will be appreciated by those skilled in the art, the number of pI variants to be included on each or both monomers to obtain good isolation will depend in part on the starting pI of the scFv and Fab of interest. That is, to determine which monomer is to be engineered or the "direction" within it (e.g., more electropositive or more electronegative), the Fv sequences of the two target antigens are calculated and a decision is made therefrom. As is known in the art, different fvs will have different starting pis that are utilized in the present invention. Typically, the pis are engineered such that the total pI difference per monomer is at least about 0.1logs, as outlined herein, preferably 0.2-0.5.

Furthermore, as will be understood by those skilled in the art and as outlined herein, heterodimers can be separated from homodimers based on size. For example, as shown in fig. 1 and 2, heterodimers with two scfvs can be isolated by the "triplet F" format and those of bispecific mabs. This can be further exploited at higher prices using additional antigen binding sites. For example, as shown otherwise, one monomer would have two Fab fragments and the other monomer would have one scFv, creating a difference in size and hence molecular weight.

In addition, as will be understood by those skilled in the art and as outlined herein, the formats outlined herein may be extended to also provide trispecific and tetraspecific antibodies. In this embodiment, some variations of which are depicted in FIG. 1A, it is recognized that it is possible for some antigens to bind divalent (e.g., two antigen binding sites bind to a single antigen; e.g., A and B can be part of a typical divalent association, and C and D can optionally be present and optionally be the same or different). It is understood that any combination of Fab and scFv can be utilized to obtain the desired results and combinations.

By using the constant region of the heavy chain, a more modular way of designing and purifying multispecific proteins, including antibodies, is provided in cases where the pI variant is used to achieve heterodimerization. Thus, in some embodiments, heterodimerization variants (including skewing and purification of heterodimerization variants) are not included in the variable regions, such that each individual antibody must be engineered. In addition, in some embodiments, the likelihood of immunogenicity caused by pI variants is significantly reduced by importing pI variants from different IgG isotypes such that the pI is altered without introducing significant immunogenicity. Thus, an additional problem to be solved is to elucidate low pI constant domains with high human sequence content, e.g., to minimize or avoid non-human residues at any particular position.

The side benefit of this pI engineering that can occur is also the prolonged serum half-life and increased FcRn binding. That is, as described in USSN 13/194,904 (incorporated by reference in its entirety), decreasing the pI of antibody constant domains, including those found in antibodies and Fc fusions, can result in longer serum retention in vivo. These pI variants for increasing serum half-life also contribute to pI changes for purification.

In addition, it should be noted that pI variants of heterodimerization variants provide additional benefits to the analytical and quality control processes of bispecific antibodies, particularly in the case of CD3 antibodies, the ability to eliminate, minimize, and distinguish when homodimers are present is significant. Similarly, the ability to reliably detect the reproducibility of heterodimeric protein production is important.

In addition to all or part of the variant heavy chain constant domain, one or both monomers may contain one or two fusion partners, such that the heterodimer forms a multivalent protein. As generally depicted in the figures, and in particular fig. 1A, the fusion partners are depicted as A, B, C and D, including all possible combinations. Typically, A, B, C and D are selected such that the heterodimer is at least bispecific or bivalent in its ability to interact with another protein.

As will be appreciated by those skilled in the art and discussed more fully below, the heterodimeric fusion proteins of the present invention can assume a number of configurations, as generally depicted in fig. 1 and 2. Some figures depict "single-ended" configurations in which one type of specificity is present on one "arm" of the molecule and a different specificity is present on the other "arm". Other figures depict "double-ended" configurations in which there is at least one type of specificity at the "top" of the molecule and one or more different specificities at the "bottom" of the molecule. Furthermore, as shown, these two configurations can be combined, where three or four specificities can exist based on the particular combination. Accordingly, the present invention provides "multispecific" binding proteins, including multispecific antibodies. Thus, the present invention relates to novel immunoglobulin compositions that co-engage at least a first and a second antigen. The first and second antigens of the invention are referred to herein as antigen-1 and antigen-2, respectively.

One heterodimeric backbone of particular utility in the present invention is the "F triplet" or "bottle opener" backbone form. In this embodiment, one heavy chain of the antibody comprises a single chain Fv ("scFv", as defined below) and the other heavy chain is in the form of a "regular" FAb comprising variable heavy and light chains. This structure is sometimes referred to herein as an "F triplet" format (scFv-FAb-Fc) or a "bottle-opener" format because the rough image resembles a bottle opener (see fig. 1B). The two chains are joined together through the use of amino acid variants in the constant region (e.g., Fc domain and/or hinge region) that promote the formation of heterodimeric antibodies as described more fully below.

There are several distinct advantages to the "F triplet" form of the invention. As known in the art, antibody analogs that rely on two scFv constructs often have stability and aggregation problems that can be mitigated in the present invention by adding "regular" heavy and light chain pairings. In addition, there is no problem of incorrect pairing of heavy and light chains (e.g., heavy chain 1 is paired with light chain 2, etc.), as opposed to formats that rely on two heavy chains and two light chains.

In addition to all or part of the variant heavy chain constant domain, one or both monomers may contain one or two fusion partners, such that the heterodimer forms a multivalent protein. As generally depicted in fig. 64 of USSN 13/648,951 (the legend accompanying which is incorporated herein by reference), fusion partners are depicted as A, B, C and D, including all possible combinations. Typically, a, B, C and D are selected such that the heterodimer is at least bispecific or bivalent in its ability to interact with another protein. In the case of the "F triplet" form of the invention, typically A and B are scFv and Fv (it being understood that either monomer may comprise an scFv and the other may comprise an Fv/Fab), and optionally followed by one or two additional fusion partners.

Furthermore, as outlined herein, additional amino acid variants may be introduced into the bispecific antibodies of the invention to add additional functionality. For example, amino acid changes within the Fc region (added to one or both monomers) can be added to promote increased ADCC or CDC (e.g., altered binding to Fc γ receptors); allowing or increasing the yield of added toxins and drugs (e.g., for ADCs), as well as increasing binding to FcRn and/or increasing the serum half-life of the resulting molecule. As further described herein and as will be understood by one of skill in the art, any and all of the variants outlined herein may optionally and independently be combined with other variants.

Similarly, another class of functional variants is "Fc γ abrogation variants" or "Fc knockout (FcKO or KO) variants. In these embodiments, for some therapeutic applications, it is desirable to reduce or remove normal binding of the Fc domain to one or more or all of the Fc γ receptors (e.g., Fc γ R1, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa, etc.) to avoid other mechanisms of action. That is, for example, in many embodiments, particularly in the use of bispecific antibodies that bind monovalent to CD3 and another binds to a tumor antigen (e.g., CD19, her2/neu, etc.), it is often desirable to remove fcyriiia binding to eliminate or significantly reduce ADCC activity.

Definition of

In order to more fully understand the present application, some definitions are given below. These definitions are intended to include grammatical equivalents.

Herein, "elimination" means reduction or removal of activity. Thus, for example, "abrogate fcyr binding" means that an Fc region amino acid variant has less than 50% initial binding, preferably less than 70-80-90-95-98% loss of activity, compared to an Fc region that does not contain the specific variant, and typically, activity is below detectable binding levels in a Biacore assay. Particularly useful in abrogating Fc γ R binding are those shown in figure 7.

"ADCC" or "antibody-dependent cell-mediated cytotoxicity" as used herein means a cell-mediated reaction in which Fc γ R-expressing non-specific cytotoxic cells recognize bound antibody on target cells, which subsequently results in lysis of the target cells. ADCC and binding to Fc γ RIIIa; increased binding to Fc γ RIIIa results in increased ADCC activity.

"ADCP" or antibody-dependent cell-mediated phagocytosis, when used herein, means a cell-mediated reaction in which Fc γ R-expressing non-specific cytotoxic cells recognize bound antibody on target cells, which subsequently results in phagocytosis of the target cells.

By "modification" is meant herein amino acid substitutions, insertions, and/or deletions in the polypeptide sequence or alterations to the moiety chemically linked to the protein. For example, the modification may be an altered sugar or PEG structure attached to the protein. By "amino acid modification" is meant herein amino acid substitutions, insertions, and/or deletions in the polypeptide sequence. For clarity, unless otherwise noted, amino acid modifications are always the amino acids encoded by DNA, e.g., 20 amino acids with codons in DNA and RNA.

By "amino acid substitution" or "substitution" is meant herein the substitution of a different amino acid for the amino acid at a particular position in the parent polypeptide sequence. In particular, in some embodiments, the substitution is not a naturally occurring amino acid at the particular position, nor is it a naturally occurring amino acid within the organism or any organism. For example, the substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced by tyrosine. For clarity, proteins that have been engineered to alter the nucleic acid coding sequence without altering the starting amino acids (e.g., exchange of CGG (encoding arginine) for CGA (still encoding arginine) to increase host organism expression levels) are not "amino acid substitutions"; that is, although a new gene encoding the same protein is formed, if the protein has the same amino acid at a specific position where it starts, it is not an amino acid substitution.

"amino acid insertion" or "insertion" when used herein means the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, -233E or 233E means that the glutamic acid is inserted after position 233 and before position 234. Alternatively, -233ADE or a233ADE means that alaasp glu is inserted after position 233 and before position 234.

"amino acid deletion" or "deletion" when used herein means the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, E233-or E233# or E233() refers to the deletion of the glutamic acid at position 233. In addition, EDA 233-or EDA233# refers to the deletion of the sequence GluAspAla at position 233.

A "variant protein" or "protein variant", or "variant", as used herein, means a protein which differs from the parent protein by at least one amino acid modification, a protein variant may refer to a protein per se, a composition comprising the protein, or an amino acid sequence encoding the same, preferably a protein variant having at least one amino acid modification as compared to the parent protein, e.g., about 1 to about 70 amino acid modifications as compared to the parent protein, and preferably about 1 to about 5 amino acid modifications, as described below, in some embodiments the parent polypeptide, e.g., Fc parent polypeptide, is a human wild-type sequence, such as an Fc region from IgG1, IgG2, IgG 585 or IgG4, although a human sequence having a variant may also serve as a "parent polypeptide", e.g., IgG1/2 cross-linked to the protein of FIG. 13, a protein variant sequence herein will preferably have at least about 80% identity, most preferably at least about 90% identity, more preferably at least about 95-98-99% identity, or more preferably at least about the amino acid sequence of the amino acid variant of the parent protein sequence as mentioned herein, or the amino acid variant of the amino acid modification of the amino acid variant of the polypeptide of the invention, such as described in the invention under the invention by the invention under the invention, such as described in the invention under the entire specification WO 3883.7/specification, the invention under the invention, the entire specification of the invention under the specification of the invention, the invention under the specification of the invention under the patent specification of the invention, the patent specification of the invention under the patent specification of the invention under the specification of the invention, the invention under the patent No. WO 417/patent No. 7, the invention under the invention, the invention under the invention, the invention under the specification of the invention under the patent No. the invention under the specification of the invention under the specification of the patent No. the invention under the patent No. the invention under the patent No. the invention under the patent No. the invention under the patent No. where the invention under the patent No. where the invention under the patent No. the invention under the patent No. where the invention under the patent No. where the patent No. where the patent No. WO 7/No. the patent No. where the patent No. where the patent No. 7/No. where the patent No. where the invention the patent No. where.

As used herein, "protein" means herein at least two covalently linked amino acids including proteins, polypeptides, oligopeptides and peptides peptidyl may include naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e., "analogs", such as peptoids (see Simon et al, PNAS USA 89(20):9367(1992), incorporated by reference in their entirety) amino acids may be naturally occurring or synthetic (e.g., amino acids not encoded by DNA), as understood by those skilled in the art. for example, homo-phenylalanine, citrulline, ornithine and norleucine are considered synthetic amino acids for the purposes of the present invention and may utilize both D-and L- (R or S) conformations amino acids.variants of the present invention may include modifications comprising synthetic amino acids incorporated using techniques developed, for example, by Schultz and colleagues, including but not limited to the synthetic amino acids incorporated by Cropp & Shutz, 2004, Andes Genet.20(12): Trenson-30, 2003, et al, USA, 2000-7, Pro-9, Ser. 7, Ser. No.: 92, Ser. 2, Ser. No.: 7, No.: schematic, No.: 7, No.: schematic, No.: see, No.: schematic, 7, et al, incorporated by reference in which includes references therein, incorporated by reference to a synthetic amino acid, et al, (7, et al).

"residue" when used herein means a position in a protein and its associated amino acid identity. For example, asparagine 297 (also referred to as Asn297 or N297) is the residue at position 297 in human antibody IgG 1.

"Fab" or "Fab region" when used herein means a polypeptide comprising VH, CH1, V L and C L immunoglobulin domains Fab may refer to this region in isolation, or in the case of a full-length antibody, antibody fragment or Fab fusion protein.

"IgG subclass modification" or "isotype modification" as used herein means amino acid modifications that convert one amino acid of one IgG isotype to the corresponding amino acid in a different, aligned IgG isotype. For example, since IgG1 contains tyrosine and IgG2 contains phenylalanine at EU position 296, the F296Y substitution in IgG2 is considered an IgG subclass modification.

"non-naturally occurring modification" when used herein means a modification of an amino acid that is not of the same type. For example, since none of the iggs contained a serine at position 434, substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) was considered a non-naturally occurring modification.

"amino acid" and "amino acid identity" as used herein means one of the 20 naturally occurring amino acids encoded by DNA and RNA.

"effector function" as used herein means a biochemical event caused by the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP and CDC.

By "IgG Fc ligand" as used herein is meant a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc/Fc ligand complex. Fc ligands include, but are not limited to, Fc γ RIs, Fc γ RIIs, Fc γ RIIIs, FcRn, C1q, C3, mannose binding lectin, mannose receptor, staphylococcal protein a, streptococcal protein G, and viral Fc γ R. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors homologous to Fc γ R (Davis et al, 2002, Immunological Reviews 190: 123-. Fc ligands may include molecules that bind Fc not found. Specific IgG Fc ligands are FcRn and Fc γ receptors. "Fc ligand" as used herein means a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc/Fc ligand complex.

In humans, this family includes, but is not limited to, Fc γ RI (CD64), including isoforms Fc γ RIa, Fc γ RIb, and Fc γ RIc, Fc γ RII (CD32), including isoforms Fc γ RIIa (including allotype H131 and R131), Fc γ RIIb (including Fc γ RIIb-1 and Fc γ RIIb-2), and Fc γ RIIc, and Fc γ RIII (CD16), including isoforms Fc γ RIIIa (including allotype V158 and F158), and Fc γ RIIb (including allotype Fc γ RIIb-NA1 and Fc γ RIIb-NA2) (Jefferis et al, 2002, im L ett: 57-65, incorporated by reference in its entirety), and any undesidered human γ RIIb or Fc γ RIIb-NA2) (Jefferis et al, 2002, im L ett: 57-65, and any undefound human γ Fc γ R or Fc γ rir L ett, and Fc γ rir 3526, and mouse Fc γ rirs), and mouse Fc γ RIII (CD 3826, Fc γ RI 2), and Fc γ RIII).

As known in the art, a functional FcRn protein comprises two polypeptides, often referred to as a heavy chain and a light chain, the light chain is β -2-microglobulin and the heavy chain is encoded by the FcRn gene unless otherwise noted herein, FcRn or FcRn protein refers to the FcRn heavy chain in complex with β -2-microglobulin, various FcRn variants used to increase binding to the FcRn receptor and in some cases increase serum half-life are shown in the figure legend of fig. 83.

"parent polypeptide" as used herein means a starting polypeptide that is subsequently modified to produce a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered form of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or an amino acid sequence encoding the same. Thus, "parent immunoglobulin" when used herein means an unmodified immunoglobulin polypeptide modified to produce a variant, and "parent antibody" when used herein means an unmodified antibody modified to produce a variant antibody. It should be noted that "parent antibody" includes known commercially available, recombinantly produced antibodies as outlined below.

By "Fc fusion protein" or "immunoadhesin" is meant herein a protein comprising an Fc region, which is typically linked (optionally through a linker moiety, as described herein) to a different protein, such as a binding moiety to a target protein, as described herein. In some cases, one monomer of the heterodimeric protein comprises an antibody heavy chain (comprising an scFv or further comprising a light chain) and the other monomer is an Fc fusion, comprising a variant Fc domain and a ligand. In some embodiments, these "half antibody-half fusion proteins" are referred to as "fusions".

"position" as used herein means a location in the sequence of a protein. Positions may be numbered consecutively, or numbered according to established formats, such as the EU index for antibody numbering.

"target antigen" as used herein means a molecule that is specifically bound by the variable region of a given antibody. The target antigen may be a protein, carbohydrate, lipid, or other chemical compound. A large number of suitable target antigens are described below.

"Strandedness" in the context of the monomers of the heterodimeric proteins of the present invention means: similar to the two strands of "matched" DNA (strand), heterodimeric variants are incorporated into each monomer so as to retain the ability to "match" to form heterodimers. For example, if some pI variants are engineered into monomer a (e.g., to make the pI higher), then these are "charge pairs" that can be exploited and sterically hindered variants that do not interfere with the pI variants, e.g., the charge variants that make the pI higher are placed on the same strand or "monomer" to retain both functionalities.

"target cell" as used herein means a cell that expresses a target antigen.

By "variable region" as used herein is meant an immunoglobulin region comprising one or more Ig domains, which is substantially encoded by any of the v.kappa., v.lamda., and/or VH genes constituting the kappa, lambda, and heavy chain immunoglobulin loci, respectively.

"wild type or WT" means herein an amino acid sequence or a nucleotide sequence as found in nature, including allelic variations. The WT protein has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.

The antibodies of the invention are typically isolated or recombinant. "isolated" when used to describe the various polypeptides disclosed herein means a polypeptide that has been identified and isolated and/or recovered from a cell or cell culture in which it is expressed. Typically, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities.

By "specifically binds" or "specifically binds to" or "specific for" a particular antigen or epitope is meant a measurable binding that is distinct from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a molecule having a similar structure but no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

Specific binding to a particular antigen or epitope can be manifested, for example, by an antibody having a KD for the antigen or epitope of at least about 10-4M, at least about 10-5M, at least about 10-6M, at least about 10-7M, at least about 10-8M, at least about 10-9M, alternatively at least about 10-10M, at least about 10-11M, at least about 10-12M, or higher, wherein KD refers to the dissociation rate of the particular antibody-antigen interaction. Typically, the antibody that specifically binds an antigen will have a KD that is 20-,50-,100-,500-,1000-,5,000-,10,000-or more-fold greater than the control molecule for the antigen or epitope.

In addition, specific binding to a particular antigen or epitope can be manifested, for example, as an antibody having a KA or KA to the antigen or epitope that is at least 20-,50-,100-,500-,1000-,5,000-,10,000-or more times greater relative to a control epitope, where KA or KA refers to the off-rate of a particular antibody-antigen interaction.

Heterodimeric proteins

The present invention relates to the generation of multispecific, in particular bispecific, binding proteins, and in particular, multispecific antibodies. The present invention generally relies on the use of engineered or variant Fc domains that can self-assemble in a producer cell to produce heterodimeric proteins, as well as methods of producing and purifying such heterodimeric proteins.

Antibodies

In general, the term "antibody" includes any polypeptide comprising at least one constant domain, including but not limited to CH1, CH2, CH3, and C L.

Conventional antibody building blocks typically comprise tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light chain" (typically having a molecular weight of about 25 kDa) and one "heavy chain" (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The present invention relates to IgG classes, which have several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG 4. Thus, "isotype" as used herein means any subclass of immunoglobulin defined by the chemical and antigenic properties of its constant regions. It is understood that therapeutic antibodies may also include hybrids of the isotype and/or subclass. For example, pI engineering of the IgG1/G2 hybrid is encompassed by the present invention as shown in US publication No. 2009/0163699 (incorporated by reference).

The amino-terminal portion of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition and is commonly referred to in the art and herein as the "Fv domain" or "Fv region". In the variable region, each V domain to the heavy and light chains aggregates into three loops to form an antigen binding site. Each loop is called a complementarity determining region (hereinafter referred to as "CDR"), in which the change in amino acid sequence is most significant. "variable" refers to the fact that: the sequences of certain segments of the variable regions vary widely between antibodies. The variability within the variable zone is not evenly distributed. Instead, the V region is composed of relatively invariant stretches of 15-30 amino acids, called Framework Regions (FRs), separated by short regions of extreme variability, called "hypervariable regions", each of 9-15 amino acids or longer in length.

Each VH and V L is composed of three hypervariable regions ("complementarity determining regions", "CDRs") and 4 FRs, arranged from amino-terminus to carboxy-terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

The hypervariable region typically comprises about amino acid residues 24-34 (L CDR 1; "L" refers to the light chain), 50-56 (L CDR2) and 89-97 (L CDR3) from the light chain variable region and about 31-35B (HCDR 1; "H" refers to the heavy chain), amino acid residues around 50-65(HCDR2) and 95-102(HCDR 3); Kabat et al, SEQ ENCES OF PROTEINSOF IMMU L OGICA L INTEREST (sequence OF proteins OF immunological INTEREST), 5 th edition, Public HealthService, National Institutes OF Healthh, Bethesda, Md. (1991) and/or those residues in the light chain variable region that form hypervariable loops (e.g., residues 26-32 (L CDR1), 50-52 (L2) and 91-96 (91-96) (1982) and/or those in the light chain variable region that form hypervariable loops (e.g., CDR 26-32; CDR 8424 CDR 8442), 50-52 (L2) and 91-96 (1982) and about the invention described herein below: CDR 26-26.8653; CDR 60: 70 H.51-8226, and 8653) from the heavy chain variable region OF the invention.

Throughout this specification, the Kabat numbering system (broadly, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) and the EU numbering system for the Fc region are generally used when referring to residues in the variable domain (e.g., Kabat et al, supra (1991)).

The CDRs facilitate the formation of the antigen binding site (or more specifically, the epitope binding site) of the antibody. An "epitope" refers to a determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as an antibody determinant. Epitopes are aggregates of molecules such as amino acids or sugar side chains and generally have specific structural properties, as well as specific charge characteristics. A single antigen may have more than one epitope.

An epitope may comprise amino acid residues directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues not directly involved in binding, such as amino acid residues effectively blocked by a specific antigen binding peptide; in other words, the amino acid residue is within the footprint of the specific antigen-binding peptide.

Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes produced by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes can be distinguished as loss of binding to the former, but not to the latter, in the presence of denaturing solvents.

Epitopes typically comprise at least 3 amino acids, and more usually, at least 5 or 8-10 amino acids, in a unique spatial conformation. Antibodies recognizing the same epitope can be validated in simple immunoassays that show the ability of one antibody to block the binding of another antibody to the target antigen, e.g., "competition".

Based on the degree OF conservation OF the SEQUENCES, they classified the individual primary SEQUENCES as CDR and framework SEQUENCES, and tabulated them (see SEQUENCES OF IMMUNO L OGICA L INTEREST, 5 th edition, NIH publication, Nos. 91-3242, E.A. Kabat et al, incorporated by reference in their entirety).

In the IgG subclass of immunoglobulins, several immunoglobulin domains are present in the heavy chain. By "immunoglobulin (Ig) domain" is meant herein an immunoglobulin region with a distinct tertiary structure. Of interest in the present invention are heavy chain domains, including the constant heavy Chain (CH) domain and the hinge domain. In the case of IgG antibodies, the IgG isotypes each have three CH regions. Thus, in the case of IgG the "CH" domains are as follows: according to the EU index "CH 1" as in Kabat refers to positions 118- > 220. Position 237-. As shown herein and described below, pI variants can be in one or more CH regions, as well as hinge regions, as discussed below.

It should be noted that the sequence depicted herein begins in region CH1, position 118; except as noted, the variable regions are not included. For example, the first amino acid of SEQ ID NO2, denoted as position "1" in the sequence Listing, corresponds to position 118 of the CH1 region, according to EU numbering rules.

Another type of Ig domain of the heavy chain is the hinge region. By "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" is meant herein a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, while the IgG CH2 domain begins at residue EU position 237. Thus for IgG, an antibody hinge is defined herein as comprising positions 221 (D221 in IgG 1) to 236 (G236 in IgG 1), wherein the numbering convention is according to the EU index as in Kabat. In some embodiments, for example, in the case of an Fc region, a lower hinge is included, "lower hinge" generally refers to position 226 or 230. As mentioned herein, pI variants can also be made in the hinge region.

The light chain typically comprises two domains, a variable light domain (containing the light chain CDRs and a variable heavy domain that forms an Fv region) and a constant light region (often referred to as C L or ck).

Another region of interest for other substitutions is the Fc region, outlined below. "Fc" or "Fc region" or "Fc domain" when used herein means a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain, and in some cases comprising part of a hinge. Fc thus refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, the Fc may comprise a J chain. For IgG, the Fc domain includes the immunoglobulin domains C γ 2 and C γ 3(C γ 2 and C γ 3) and the lower hinge region between C γ 1(C γ 1) and C γ 2(C γ 2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined to include residues C226 or P230 to its carboxy terminus, with the numbering convention being in accordance with the EU index as in Kabat. In some embodiments, amino acid modifications are made to the Fc region, e.g., to alter binding to one or more fcyr receptors or to FcRn receptors, as described more fully below.

Thus, in some embodiments, the invention provides heterodimeric antibodies that rely on the use of two different heavy chain variable Fc domains that will self-assemble to form the heterodimeric antibody.

In some embodiments, the antibody is full-length. By "full length antibody" is meant herein a structure that constitutes the natural biological form of an antibody, including the variable and constant regions, comprising one or more modifications as outlined herein, particularly in the Fc region, to allow heterodimerization to form or purify heterodimers separate from homodimers. Full-length heterodimeric antibodies are two heavy chains and two light chains or a common light chain with different Fc domains.

Alternatively, antibodies may include a variety of structures as shown generally in the figures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies (minibodies), domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and separately respective fragments.

In one embodiment, antibodies are antibody fragments so long as they contain at least one constant domain that can be engineered to produce heterodimers (such as pI engineering). other antibody fragments that can be used include fragments containing one or more of the CH1, CH2, CH3, hinge, and C L domains of the invention that have been pI engineered.

scFv embodiments

In some embodiments of the invention, one monomer comprises a heavy chain comprising a scFV linked to an Fc domain, and another monomer comprises a heavy chain comprising a Fab linked to an Fc domain, e.g., a "classical" heavy chain, and a light chain, "Fab" or "Fab region," as used herein, means a polypeptide comprising VH, CH1, V L, and C L immunoglobulin domains.

Several of the heterodimeric antibody embodiments described herein rely on the use of one or more scFv domains, comprising a variable heavy chain and a variable light chain, covalently linked using a linker, to form an antigen binding domain. Some embodiments use "standard" linkers herein, typically glycine and serine linkers, as are well known in the art.

The invention further provides a charged scFv linker to facilitate pI separation between the first and second monomers. That is, by adding a charged scFv linker, either positively or negatively charged (or both, in the case of using the backbone of the scFv on a different monomer), this allows the monomer comprising the charged linker to alter the pI without further manufacturing changes in the Fc domain. These charged linkers can be substituted into any scFv containing a standard linker. Again, as will be appreciated by those skilled in the art, a charged scFv linker is used on the correct "chain" or monomer, depending on the pI change desired. For example, as discussed herein, to make the F triplet form heterodimeric antibody, the original pI of the Fv region of each desired antigen binding domain is calculated, one of the making scfvs is selected, and depending on the pI, either the positively or negatively charged structure is selected.

In addition, in the case of the anti-CD 3 scFv region, disulfide bonds can be engineered into the variable heavy and variable light chains to provide additional stability. Suitable disulfide sequences in the context of anti-CD 3 scFv are shown in figure 8.

Chimeric and humanized antibodies

In some embodiments, the antibody may be a mixture from different species, e.g.a chimeric antibody and/or a humanized antibody, typically the "chimeric antibody" and the "humanized antibody" refers to an antibody which combines regions from more than one species, e.g.the "chimeric antibody" conventionally comprises a variable region(s) from a mouse (or in some cases a rat) and a constant region(s) from a human being, e.g.the "humanized antibody" typically refers to a non-human antibody which has a variable domain framework region exchanged for sequences present in a human antibody, typically in a humanized antibody which is encoded by a polynucleotide of human origin or which is identical to a nucleic acid derived from a non-human organism apart from the CDRs in addition to the CDRs in which some or all of the CDRs are encoded by a nucleic acid of a human antibody which is grafted into a β -sheet framework of a human variable region to form an antibody which specificity is determined by the formation of the grafted human antibody, e.g.g.A. the antibody as cited in e.g.g.7. the human Antibodies as the parent sequence of the invention is cited in e.g.35. the invention, USA, the invention is preferably the invention, the invention is described in the invention, the invention is described in the methods of the invention, the invention is described in the invention, the methods of the invention are described in the invention, the methods of the Antibodies are described in the invention, the methods of the invention, the Antibodies are described in the invention, the methods of the invention, the methods of the invention, the methods of the invention, the methods of the invention, the use of the invention, the methods of the use of the invention, the methods of the invention, the invention are described by the invention, the invention are described by the invention, the methods of the.

Heterodimeric heavy chain constant regions

Thus, based on the use of a monomer containing a variant heavy chain constant region, and in particular an Fc domain, as the first domain, the present invention provides heterodimeric proteins. By "monomer" is meant herein one half of a heterodimeric protein. It should be noted that traditional antibodies are in fact tetramers (two heavy chains and two light chains). In the context of the present invention, a pair of heavy-light chains (if applicable, e.g., if the monomer comprises a Fab) is considered to be a "monomer". Similarly, the heavy chain region comprising the scFv is considered a monomer. In the case where the Fv region is one fusion partner (e.g., heavy and light chain variable domains) and the non-antibody protein is the other fusion partner, each "half" is considered a monomer. Essentially, each monomer contains enough heavy chain constant region to allow heterodimeric engineering, whether all constant regions, e.g., Ch 1-hinge-Ch 2-Ch3, Fc region (Ch2-Ch3), or just Ch3 domain.

The variant heavy chain constant region may comprise all or part of a heavy chain constant region, including the full length construct, CH 1-hinge-CH 2-CH3, or portions thereof, including, for example, CH2-CH3 or CH3 alone. In addition, the heavy chain region of each monomer may be the same backbone (CH 1-hinge-CH 2-CH3 or CH2-CH3) or a different backbone. N-and C-terminal truncations and lengtheners are also included within the definition; for example, some pI variants include the addition of charged amino acids to the C-terminus of the heavy chain domain.

Thus, typically, one monomer of the "triple F" constructs of the invention is the scFv region-hinge-Fc domain) and the other is (VH-CH 1-hinge-CH 2-CH3 plus the associated light chain), with heterodimerization variants, including steric variants, isotype variants, charge-steering, and pI variants, Fc and FcRn variants, elimination variants, and other antigen binding domains (with optional linkers) included in these regions.

In addition to the heterodimerization variants outlined herein (e.g., steric hindrance and pI variants), the heavy chain region may also contain additional amino acid substitutions, including changes for altering fcyr and FcRn binding as discussed below.

In addition, some monomers may utilize a linker between the variant heavy chain constant region and the fusion partner. For the scFv portion of the "bottle opener," standard linkers known in the art, or charged scFv linkers described herein, can be used. In cases where additional fusion partners are prepared (e.g., FIGS. 1 and 2), conventional peptide linkers can be used, including flexible linkers of glycine and serine, or the charged linker of FIG. 9. In some cases, the linkers used as components of the monomers are different from those defined below for the ADC constructs, and in many embodiments are not cleavable linkers (such as those susceptible to proteases), although cleavable linkers may be used in some embodiments.

Heterodimerization variants include many different types of variants, including, but not limited to, steric variants (including charge variants) and pI variants, which may optionally and independently be combined with any other variant. In these embodiments, it is important to match "monomer a" with "monomer B"; that is, if the heterodimeric protein is dependent on both steric and pI variants, these need to be matched correctly to each monomer: for example, a functional set of sterically hindered variants (1 on monomer a,1 on monomer B) is combined with a set of pI variants (1 on monomer a,1 on monomer B) such that the variants on each monomer are designed to perform the desired function, keeping the pI "chained" such that sterically hindered variants that can alter the pI are placed on the appropriate monomer.

It is important to note that heterodimerization variants outlined herein (e.g., including but not limited to those shown in fig. 3 and 12) can be optionally and independently combined with any other variant, and on any other monomer. That is, it is important for heterodimerization that there be "multiple sets" of variants, one for one monomer and one for the other. Whether these are combined together from FIG. 1 to 1 (e.g.the list of monomers 1 can be matched) or switched (replacement of the monomer 1pI variant with the monomer 2 hindered variant) is irrelevant. However, as described herein, the "chain" should be retained when combined as outlined above. In addition, for other Fc variants (such as for Fc γ R binding, FcRn binding, etc.), independently and optionally, either monomer, or both monomers, can include any of the enumerated variants. In some cases, two monomers have additional variants, and in some cases only one monomer has additional variants, or they may be combined together.

Heterodimerization variants

The present invention provides heterodimeric proteins, including various forms of heterodimeric antibodies, that utilize heterodimeric variants to allow heterodimer formation and/or purification separate from homodimers.

Sterically hindered variants

In some embodiments, heterodimer formation may be facilitated by the addition of steric variants. That is, by changing the amino acids in each heavy chain, it is more likely that different heavy chains associate to form a heterodimeric structure rather than forming a homodimer with the same Fc amino acid sequence. Suitable steric hindrance variants are included in fig. 3 and fig. 12A, 12B, 12C, 12D, 12F and 12G.

One mechanism, commonly referred to in the art as "knobs and holes", refers to the engineering of amino acids to create steric hindrance effects to favor heterodimer formation and may optionally also use amino acid engineering to disfavor homodimer formation; this is sometimes referred to as "knobs and holes" as in USSN 61/596,846, Ridgway et al, Protein Engineering 9(7):617 (1996); atwell et al, J.mol.biol.1997270: 26; all documents described in US patent No. 8,216,805 are incorporated herein by reference in their entirety. The figures identify a number of "monomer a-monomer B" pairs, which rely on "knobs and holes". In addition, these "knob and hole" mutations can be combined with disulfide bonds to bias formation towards heterodimerization as described in Merchant et al, Nature Biotech.16:677 (1998).

One other mechanism useful in the generation of heterodimers is sometimes referred to as "electrostatic steering," as described in Gunasekaran et al, j.biol.chem.285(25):19637(2010), which is incorporated herein by reference in its entirety, this is sometimes referred to herein as "charge pairing.

Additional monomer a and monomer B variants that can be combined with other variants, optionally and independently in any amount, such as pI variants as outlined herein or other sterically hindered variants shown in figure 37 of US 2012/0149876, the figures and legends of which and SEQ ID NOs are specifically incorporated herein by reference.

In some embodiments, the sterically hindered variants outlined herein may optionally and independently bind to any pI variant (or other variants such as Fc variants, FcRn variants, etc.) in one or both monomers, and may or may not be independently and optionally included by the proteins of the invention.

pI (isoelectric point) variants for heterodimers

Generally, as will be appreciated by those skilled in the art, there are two broad classes of pI variants: those that increase the pI of the protein (basic changes) and those that decrease the pI of the protein (acidic changes). As described herein, all combinations of these variants can be made: one monomer may be wild type or a variant that does not exhibit a significantly different pI than wild type, and the other may be more basic or acidic. Alternatively, each monomer is changed, one to become more basic and one to become more acidic.

preferred combinations of pI variants are shown in fig. 3 and 12E.

Heavy chain pI alteration

A number of pI variants are shown in figures 54 and 55. As outlined herein and shown in the figures, these changes are shown relative to IgG1, but all isotypes can be changed in this manner, as well as isotype hybrids. In the case where the heavy chain constant domain is from IgG2-4, R133E and R133Q may also be used.

Antibody heterodimeric light chain variants

In the case of antibody-based heterodimers, for example where at least one monomer comprises a light chain in addition to a heavy chain domain, pI variants can also be made in the light chain. Amino acid substitutions for reducing the pI of the light chain include, but are not limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E, K207E and the addition of the peptide DEDE at the c-terminus of the light chain. Such changes based on constant λ light chains include one or more substitutions at R108Q, Q124E, K126Q, N138D, K145T and Q199E. In addition, the pI of the light chain can also be increased.

Isoform variants

In addition, many embodiments of the invention rely on "importing" pI amino acids at specific positions from one IgG isotype into another IgG isotype, thereby reducing or eliminating the possibility of unwanted immunogenicity being introduced into the variants. Many of these are shown in fig. 10A and 10B. That is, IgG1 is a common isotype used for therapeutic antibodies for a variety of reasons, including high effector function. However, the pI of the heavy chain constant region of IgG1 was higher than IgG2(8.10 versus 7.31). By introducing the IgG2 residue at a specific position into the IgG1 backbone, the pI of the resulting monomer is reduced (or increased) and additionally exhibits a longer serum half-life. For example, IgG1 has a glycine at position 137 (pI 5.97), IgG2 has a glutamic acid (pI 3.22); glutamate input will affect the pI of the resulting protein. As described below, many amino acid substitutions are typically required to significantly affect the pI of a variant antibody. However, as discussed below, it should be noted that equal variation of the IgG2 molecules increases serum half-life.

In other embodiments, non-isoform amino acid changes are made to reduce the overall charge state of the resulting protein (e.g., by changing a higher pI amino acid to a lower pI amino acid), or to adapt the structure for stability, etc., as described further below.

In addition, significant changes in each monomer of the heterodimer can be seen by engineering the heavy and light chain constant domains with pis. As discussed herein, differing the pI of two monomers by at least 0.5 can allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point.

Calculating pI

The pI of each monomer may depend on the pI of the variant heavy chain constant domain and the pI of all monomers (including the variant heavy chain constant domain and the fusion partner). Thus, in some embodiments, using the graph in fig. 53, the pI change is calculated based on the variant heavy chain constant domain. As discussed herein, the monomer to be engineered is generally determined by the Fv and the intrinsic pI of the framework regions. Alternatively, the pI of each monomer can be compared.

Heterodimeric Fc fusion proteins

In addition to the heterodimeric antibody, the invention provides a heterodimeric protein comprising a first monomer comprising a variant Fc region and a first fusion partner, and a second monomer also comprising a variant Fc region and a second fusion partner. The multiple variant Fc regions are engineered as described herein for the antibody and are thus different, and typically the first and second fusion partners are also different. In some cases, where one monomer is antibody-based (i.e., contains standard heavy and light chains or is an Fc domain with an scFv) and the other monomer is an Fc fusion protein, the resulting heterodimeric protein is referred to as a "fusion.

Better in vivo FcRn binding pI variants were also obtained

In cases where pI variants reduce the pI of a monomer, they may have additional benefits: increase the in vivo serum retention time.

Although still under examination, it is believed that the Fc region has a longer half-life in vivo, since binding to FcRn in the endosome at pH6 sequesters Fc (Ghetie and Ward,1997Immunol today.18(12):592-598, incorporated by reference in its entirety). The endosomal compartment then recirculates the Fc to the cellular epitope. Once the chamber is open to the extracellular space, the higher pH-7.4 induces Fc release back into the blood. In mice, Dall 'Acqua et al demonstrated that Fc mutants with increased binding to FcRn at pH6 and pH 7.4 actually had reduced serum concentrations and the same half-life as wild-type Fc (Dall' Acqua et al 2002, J.Immunol.169:5171-5180, incorporated by reference in its entirety). The increased affinity of Fc for FcRn at pH 7.4 is believed to prevent Fc release back into the blood. Thus, Fc mutations that would increase Fc half-life ideally increase FcRn binding at lower pH while still allowing release of Fc at higher pH. The amino acid histidine changes its charge state in the pH range of 6.0-7.4. Thus, it is not surprising that His residues are found at important positions in the Fc/FcRn complex.

In recent years, it has been shown that antibodies with variable regions having lower isoelectric points can also have longer serum half-lives (Igawa et al, 2010PEDS.23(5):385-392, incorporated by reference in its entirety). However, the mechanism of this phenomenon is not yet understood. Furthermore, the variable regions differ from antibody to antibody. Constant region variants with reduced pI and extended half-life will provide a more modular approach to improve the pharmacokinetic properties of antibodies, as described herein.

pI variants that can be used in this embodiment, as well as their use for purification optimization, are disclosed in figure 20.

Combinations of heterodimer variants

As will be understood by those skilled in the art, all of the noted heterodimerization variants can be combined optionally and independently in any manner so long as they retain their "chain" or "monomer partitioning". In addition, all of these variants can be combined in any heterodimeric form.

In the case of pI variants, although embodiments of particular utility are shown in the figures, other combinations can be made, following the basic rule of varying the pI difference between two monomers to facilitate purification.

Nucleic acid of the present invention

The invention further provides nucleic acid compositions encoding the heterodimeric proteins of the invention. As will be appreciated by those skilled in the art, the nucleic acid composition will depend on the form and backbone of the heterodimeric protein. Thus, for example, where the format requires three amino acid sequences, such as an F triplet format (e.g., a first amino acid monomer comprising an Fc domain and scFv, a second amino acid monomer comprising a heavy chain and a light chain), the three nucleic acid sequences can be incorporated into one or more expression vectors for expression. Similarly, some formats (e.g., such as the bis-scFv format disclosed in fig. 1M) require only two nucleic acids; again, they may be placed in one or two expression vectors.

Target antigens

The heterodimeric proteins of the present invention can be targeted to virtually any antigen. The "triple F" form is particularly useful for targeting two (or more) different antigens. (as outlined herein, this targeting can be any combination of monovalent and bivalent binding, depending on the format). Thus, the immunoglobulin herein preferably co-engages two target antigens, although in some cases, three or four antigens may be monovalent. The specificity of each monomer can be selected from the following list. Although the F triplet immunoglobulins described herein are particularly useful for targeting different antigens, it may be beneficial in some cases to target only one antigen. That is, each monomer may have specificity for the same antigen.

For some immune receptors, such as CD3 signaling receptors on T cells, it is critical that activation only occurs during conjugation to the co-conjugated target, since nonspecific cross-linking in the clinical setting can trigger cytokine storm and toxicity, by conjugating such antigens univalent rather than multivalent, using the immunoglobulins described herein, such activation occurs only in response to cross-linking in the microenvironment of the primary target antigen.

Virtually any antigen can be targeted by the immunoglobulins herein, including but not limited to proteins, subunits, domains, motifs, and/or epitopes belonging to the following list of target antigens: it includes both soluble factors such as cytokines and membrane-bound factors, including transmembrane receptors: 17-IA,4-1BB,4Dc, 6-keto-PGF 1a, 8-iso-PGF 2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin A, activin AB, activin B, activin C, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAMTS 9, ADAMTS, ADAMTS4, ADATS 5, addressin, aFGF, ALCAM ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, APAF-1, APE, APJ, ARCHIL, APRIL, AR-K, AR-7, alpha-1-antitrypsin, alpha-AxV/beta-1 antagonist, ANG, PARA, APAF-1, APE 632, APART, ASCID-2, ADAM-I-2, b7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, B-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2BMP-2a, BMP-3 osteogenin, BMP-4BMP-2B, BMP-5, BMP-6Vgr-1, BMP-7 (1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, B-NGF, BOK, bombesin, bone-derived neurotrophic factor (BPDE), BPDE-DNA, BTC, complement factor 3 (C), C3, C, C, C5, C, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoembryonic antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL/10, CCR, CCR, CCR, CCR, CCR, CCR, CCR, CCR, CD, CD, CD, CD, CD, CD, CD11, CD11C, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33(p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD56, CD56, CD 685123, CD56, CD56 (B56-1), CD56, CD 685123, CD137, CD138, CD140, CD146, CD147, CINN 148, CD152, CD164, CEACAM 56, CFTR, MP, Clostridium botulinum toxin, Clostridium perfringens, C56, CXCXCXCR 56, CXCR 56, CXCR 56-56, CTCXCR 56-56, 56-C56-C56, CXCR 56-56 CL 56, CTCXCR 56-56C 56, 56-56C 56, CTCXCR 56C 56, 56C 56-56C 56, CTCXCR 56C 56-56C 56, CTCXCR 56C 56, 56C 56, CTCXCR 56C 56, CTCXCR 56C 56, CTCXCR 56C, decay accelerating factor, des (1-3) -IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, deoxyribonuclease, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, Fibroblast Activation Protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, 3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, Flt-chemotactic molecule (XXC), FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3(Vgr-2), GDF-5(BMP-14, CDMP-1), GDF-6(BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15(MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR- α 1, GFR- α 2, GFR- α 3, GITR, glucagon, Glut 4, glycoprotein b/IIIa (GP b/IIIa), GM-CSF, 130, GRO 72, half-growth factor GP-HB, growth factor-HB, or GP-HB-HBP, HCMV gB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL, Hematopoietic Growth Factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3(ErbB-3), Her4(ErbB-4), Herpes Simplex Virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human myocardial myosin, Human Cytomegalovirus (HCMV), Human Growth Hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor IgA, IgE, IGF, IGF binding protein, IGF-1R, IGP, FBI, IGF-II, IGF-1, IGF-IL 1, IL-1R, IGF-IL 1, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, Interferon (INF) - α, INF- β, INF- γ, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, integrin α 2, integrin α 3, integrin α 4/β 1, integrin α 4/β 7, integrin α 5(α V), integrin α 5/β 1, integrin α 5/β 3, integrin α 6, integrin β 1, integrin β 2, interferon γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, LKn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surfactant, luteinizing agent, lymphotoxin beta receptor, dC-AM, Mac-1, MAG-AM, Mac-AM, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), C18, Mueller-inhibiting substances, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, cerebroproptidase, neurotrophin-3, -4, or-6, Neurturin, Neuronal Growth Factors (NGF), NGF-beta, NGF, NRnFn, NO, NRG Npn, NOS-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, P150, P95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, Prorelaxin (Prorelaxin), protein C, PS, PSA, PSCA, Prostate Specific Membrane Antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANSK, relaxin A-chain, relaxin B-chain, renin, Respiratory Syncytial Virus (RSV) F, RSV, SCS, SLIP 25, OPN, OSM, OX40, PSRR, SRFR 464, PSR-CAR, PSK, SRK-1, PSRR, PSK-protein, PSK-1, PSK-protein, PSK-1, PSK, PS, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell receptor α/β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF- α, TGF- β, TGF- β Pan-specific, TGF- β RI (ALK-5), TGF- β RII, TGF- β RIIb, TGF- β RIII, TGF- β 1, TGF- β 2, TGF- β 3, TGF- β 4, TGF- β 5, thrombin, thymus-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMF 2, Tmpo, TMPRSS2, TNF, TNF- α, TNF- α β, TNF- β 2, TNFac, TNF-RI, TNF-RII, TNFRSF10 (TRAIL 1Apo-2, DR), TNFRSF10 (TRAIL DR, KILLER, TRICK-2A, TRICK-B), TNFRSF10 (TRAIL DcR, LIT, TRID), TNFRSF10 (TRAIL DcR, TRUNDD), TNFRSF11 (RANK ODF, TRANCE R), TNFRSF11 (OPG OCIF, TR), TNFRSF (TWEAK R FN), TNFRSF13 (TACI), TNFRSF13 (BAFR), TNFRSF (HVATAR, HveA, LIGHT R, TR), TNFRSF (NGFR 75NTR), TNFRSF (BCMA), TNFRSF (GITR), TNFRSF (TRAIY TAJ, TRADE), TNFRSF19 (RELT), TNFRSF1 (TNFRCD 120, TNFRSF-60), TNFRSF1 (FasriBB), TNFRSF 120-80, TNFRSF (TNFRSF-80), TNFRSF (TNFRSF TXF), TNFRSF (TNFRSF-CD-1, TNFRSF-CD-III), TNFRSF (TNFRSF-CD-1, TNFRSF-CD-III), TNFRSF (TNFRSF-CD-1, TNFRSF-CD-III, TNFRSF-CD-III, TNFRSF (TNFRSF-CD-III, TNFRSF-CD (TNFRF, TNFRF (TNFRF, TNFRF, ILA), TNFRSF (DR), TNFRSF (DcTRAIL TNFRH), TNFRST (DcTRAIL TNFRRH), TNFRSF (DcTRAIL TNFRSF), TNFRSF (DR Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF (TRAIL Apo-2 ligand, TL), TNFSF (TRANCE/RANK ligand, OPG ligand), TNFSF (TWEAK Apo-3 ligand, DR ligand), TNFSF (), TNFSF13 (BAFF BLYS, TALL, THK, TNFSF), TNFSF (ligand, TNFR), SF (TL 1/VEGI), TNFSF (GITR ligand, AITR ligand, TL), TNFSF1 (TNF-a selecting in, DIF, TNFSF), TNFSF1 (TNF-b LTa, TNFSF), TNFSF (LTb TNFC, TNFR, P), TNFSF (TNFSF ligand, GP), TNFSF (CD ligand, HIGM, 154, FasFSF, TNFSF (TNF-b LTSF), TNFSF ligand, TNFSF (CD-b ligand, CD 137-CD ligand, CD-CD 153), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin-2, FGR-1(flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor hemophilia, WIF-1, WNT 52, WNT2, WNT2 WNT 72/13, WNT3, WNT A, WNT3, WNT6, XCT 465, XCT 685T A, 685T A, 685T-1, 685T A, 685T A, TWNT 685T 4, TWNT 4, 685T 4 and WNT4, 685T 4, TWNT 4, 685T 4, TWNT 4 and K4, 685T 4, TWNT 4, 685T 4, TWNT 685T 4, 685T 4, TWNT 685T 4, TWNT 4, 685T 4, TWNT 685T 4, 685T 4 and K4, TWNT 4, 685T 4 and K685T 4, TWNT 4, xedr, XIAP, XPD, and receptors for hormones and growth factors. To form a bispecific or trispecific antibody of the invention, antibodies can be prepared against any combination of these antigens; that is, each of these antigens may optionally and independently be included or excluded by multispecific antibodies according to the present invention.

Exemplary antigens that can be specifically targeted by the immunoglobulins of the present invention include, but are not limited to, CD20, CD19, Her2, EGFR, EpCAM, CD3, Fc γ RIIIa (CD16), Fc γ RIIa (CD32a), Fc γ RIIb (CD32b), Fc γ RI (CD64), Toll-like receptors (T L Rs) such as T L R4 and T L R9, cytokines such as I L-2, I L-5, I L-13, I L-12, I L-23 and TNF α, cytokine receptors such as I L-2R, chemokines, chemokine receptors, growth factors such as VEGF and HGF, and the like.

A particularly preferred combination for a bispecific antibody is an antigen-binding domain to CD3 and an antigen-binding domain to CD 19; an antigen binding domain to CD3 and an antigen binding domain to CD 33; an antigen binding domain to CD3 and an antigen binding domain to CD 38. Again, in many embodiments, the CD3 binding domain is an scFv having the exemplary sequence as depicted in the figures and/or the CD3 CDRs as outlined.

The selection of the appropriate target antigen and common target depends on the desired therapeutic use. Some targets that have proven particularly amenable to antibody therapy are those with signaling function. Other therapeutic antibodies exert their effects by blocking the signaling of the receptor by inhibiting the binding between the receptor and its cognate ligand. Another mechanism of action of therapeutic antibodies is to cause down-regulation of the receptor. Other antibodies do not act by signaling through their target antigen. The choice of a common target will depend on the detailed biology underlying the pathology of the indication being treated.

Monoclonal antibody therapy has emerged as an important treatment modality for cancer (Weiner et al, 2010, Nature Reviews Immunology 10: 317-327; Reichert et al, 2005, Nature Biotechnology 23[9]: 1073-1078; specifically incorporated herein by reference). For anti-cancer therapy, it may be desirable to target one antigen (antigen-1) while co-targeting a second antigen (antigen-2), the expression of which is restricted to cancer cells, which mediates some immune killing activity. For other therapies, it may be beneficial to target two antigens together, for example, two angiogenic factors or two growth factors, each of which are known to have some role in tumor proliferation. Exemplary common targets for oncology include, but are not limited to, HGF and VEGF, IGF-1R and VEGF, Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 and Fc γ RIIIa, CD20 and Fc γ RIIIa, Her2 and Fc γ RIIIa. The immunoglobulins of the present invention may be capable of binding to VEGF and phosphatidylserine; VEGF and ErbB 3; VEGF and PLGF; VEGF and ROBO 4; VEGF and BSG 2; VEGF and CDCP 1; VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB 3; HER-2 and BSG 2; HER-2 and CDCP 1; HER-2 and ANPEP; EGFR and CD 64; EGFR and BSG 2; EGFR and CDCP 1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD 20; CD20 and CD 74; CD20 and CD 30; CD20 and DR 4; CD20 and VEGFR 2; CD20 and CD 52; CD20 and CD 4; HGF and c-MET; HGF and NRP 1; HGF and phosphatidylserine; ErbB3 and IGF 1R; ErbB3 and IGF1, 2; c-Met and Her-2; c-Met and NRP 1; c-Met and IGF 1R; IGF1,2 and PDGFR; IGF1,2 and CD 20; IGF1,2 and IGF 1R; IGF2 and EGFR; IGF2 and HER 2; IGF2 and CD 20; IGF2 and VEGF; IGF2 and IGF 1R; IGF1 and IGF 2; PDGFRa and VEGFR 2; PDGFRa and PLGF; PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR 2; PDGFRb and c-Met; PDGFRb and EGFR; RON and c-Met; RON and MTSP 1; RON and MSP; RON and CDCP 1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR; VEGFR2 and PLGF; VEGFR2 and NRP 1; VEGFR2 and RON; VEGFR2 and DLL 4; VEGFR2 and EGFR; VEGFR2 and ROBO 4; VEGFR2 and CD 55; LPA and S1P; EPHB2 and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40 and IL 6; CD40 and IGF; CD40 and CD 56; CD40 and CD 70; CD40 and VEGFR 1; CD40 and DR 5; CD40 and DR 4; CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 and CD 40; CD80 and CD 30; CD80 and CD 33; CD80 and CD 74; CD80 and CD 2; CD80 and CD 3; CD80 and CD 19; CD80 and CD 4; CD80 and CD 52; CD80 and VEGF; CD80 and DR 5; CD80 and VEGFR 2; CD22 and CD 20; CD22 and CD 80; CD22 and CD 40; CD22 and CD 23; CD22 and CD 33; CD22 and CD 74; CD22 and CD 19; CD22 and DR 5; CD22 and DR 4; CD22 and VEGF; CD22 and CD 52; CD30 and CD 20; CD30 and CD 22; CD30 and CD 23; CD30 and CD 40; CD30 and VEGF; CD30 and CD 74; CD30 and CD 19; CD30 and DR 5; CD30 and DR 4; CD30 and VEGFR 2; CD30 and CD 52; CD30 and CD 4; CD138 and RANKL; CD33 and FTL 3; CD33 and VEGF; CD33 and VEGFR 2; CD33 and CD 44; CD33 and DR 4; CD33 and DR 5; DR4 and CD 137; DR4 and IGF1, 2; DR4 and IGF 1R; DR4 and DR 5; DR5 and CD 40; DR5 and CD 137; DR5 and CD 20; DR5 and EGFR; DR5 and IGF1, 2; DR5 and IGFR, DR5 and HER-2, and EGFR and DLL 4. Other combinations of targets include one or more members of the EGF/erb-2/erb-3 family.

Other target(s) involved in neoplastic disease that the immunoglobulins herein may bind include, but are not limited to, those selected from the group consisting of: CD, CD, CD, CD, CD, BMP, IL12, IL1, IL1, 1L, IL, INHA, TNF, TNFSF, BMP, EGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, GRP, IGF, IGF, IL12, IL1, IL 1L, INHA, TGFA, TGFB, TGFB, VEGF, CDKs, FGF, FGF, FGF, FGF, FGF, IGF1, IL, BCL, CD164, CDKN1, CDKN1, CDKN1, CDKN2, CDKN2, CDKN2, CDKN, GNRH, IGFBP, IL1, IL1, ODZ, PAWR, PLG, TGFB1I, PRKD, BRCA, CDK, CDK, CDK, CDK, CDK, CDK, CDNR 2, CDNR, ESR, ESR, ESR, FBNR, AFR, ESR, ESR, AFR, ESR, ESR, ESR, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF, FGF A, FGF, FGF, FGF, FGF, FGF, IGF1, KCNR 2, IGF, IGF1, CDK, IGF, CDNR 2, CDNR, CDK, ESR, ESR, ESR, IGF, FGF, IGF, IGF, IGF1, IGF, ESR, IGF, IGF, IGF1, IGF2, IGF, IGF, ESR, IGF2, IGF, IGF1, IGF, IGF, IGF2, IGF, etc, ESR, NR0B, NR0B, NR1D, NR1H, NR1H, NR112, NR2C, NR2C, NR2E, NR2E, NR2F, NR2F, NR3C, NR3C, NR4A, NR4A, NR5A, NR5A, NR μ L, PGR, RARB, FGF, FGF, FGF, KLK, KRT, APOC, BRCA, CHGA, CHGB, CLU, COL1A, COL6A, EGF, ERBB, ERK, FGF, FGF, FGF, FGF, FGF, FGF, FGF, PSAP, FGF, FGF, GNRH, IGF, IGF, IGF, IGFBP, IGFBP, IL12, IL1, IL 1L, IL, KLHA, KLL, KLK, KLK, SKK, TK, PAP, CLDH, PAP, CDAK, CDAKH, CDAKB, CDKA, CDAKB, CDKA, CDAKB, FGF, CDK, CDAKB, KLK, KLK, KLK, KLK, KLK, KLK, CDAKK, KLK, CDAK, KLK, PAP, CDAK, CDPAP, CDAK, CDK, CDAK, CDK, CDAK, CDK, CDAK, FGF, CDK, CDAK, CDK, CLN3, CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGE B1, GAGE C1, GGT1, GSTP1, HIP1, HUMCYT 21, IL1, K6HF, KAI1, KRT 21, MIB1, PART1, PATE, PCA 1, PIAS 1, PIK3CG, PPID, PR1, PSCA, PEPTP 6852A 1, SLC 1. mu.l, STEAP, STEAP 1, TRPC 1, ANGPT1, ANTPM 1, GF1, EGF 1, EREG, FGF1, 1 CCF 1, 1D, 1, 685, CDKN 1(p 27 Kip), CDKN 2(p 161NK 4), COL6A, CTNNB (B-catenin), CTSB (cathepsin B), ERBB (Her-2), ESR, ESR, F (TF), FOSL (FRA-1), GATA, GSN (gelsolin), IGFBP, IL2RA, IL, IL6, IL6 (glycoprotein 130), ITGA (a integrin), JUN, KLK, KRT, MAP2K (c-Jun), MKI (Ki-67), NGFB (GF), NGFR, NME (M23), PGR, PLAU (uPA), PTEN, SERPINB (mammary serine protease inhibitor), SERPINE (PAI-1), TGFA, THBS (thrombin sensitive protein-1), TIE (Tie-1), TNFRSF (glycoprotein), TNFRSF (FasL), TOP2 (TP, Ci-1), zinc (Iia), CLDN-1 (GP-1), CLDGN (AG-21P/BPN), CLUSP (AG-1), CTNN protein (BPN), CTFB-1, CTFB (BPN, CTFB-1, CTFB (CTFB-B, CTFB-protein, CTFB-B (C-B, CTFB-B, CTP, CTF (C-B-protein, CTS (I-B, CTP) protein, CTFB (I-B (I-II) protein (I-II, CTP) protein, CTS (II, CTP, CTS (C-II, CTS (I-II), CTP) protein (II, CTS (II), CTS (C (II), CTS (II), CTP-II, CTP (C-II), and CTS (I (II) protein (II, CTP (II) protein (I-II), CTP (II), and CTP (II), CTP (II, CTP (I (II) protein (II) and CTP) protein (II), CT, ERBB (Her-2), FGF, FLRT (fibronectin), GABRP (GABAa), GNAS, ID, ITGA (a integrin), ITGB (B4 integrin), KLF (GC Box BP), KRT (keratin 19), KRTHB (hair-specific type II keratin), (metallothionein-III), MUC (mucin), PTGS (COX-2), RAC (p21 Rac), S100A, SCGB1D (lipophilic B), SCGB2A (mammaglobin 2), SCGB2A (mammaglobin 1), SPRR1 (Spr), THBS, THBS, THBS, and TNFP (B), RON, c-Met, CD, DLL, PLGF, CTLA, phosphatidylserine, ROBO, CD, CD, CD, CD, CD, CD, CD, CD, CD138, CD, CD, CD137, MAPF, VEGFR, SIP, CTLA, EPHA, EPDHFR, EPDHT, EPHA, PKA, EPDHFR, PKG, PKHA, KL 2, KL-III, FLT3, PDGFR α, PDGFR β, ROR1, PSMA, PSCA, SCD1 and CD 59. To form a bispecific or trispecific antibody of the invention, antibodies can be prepared against any combination of these antigens; that is, each of these antigens may optionally and independently be included or excluded by multispecific antibodies according to the present invention.

Monoclonal antibody therapy has become an important therapeutic modality for the treatment of autoimmune and inflammatory disorders (Chan & Carter,2010, Nature Reviews Immunology 10: 301-316; Reichert et al, 2005, Nature Biotechnology 23[9]: 1073-1078; specifically incorporated herein by reference). Many proteins are generally associated with autoimmune and inflammatory responses, and thus can be targeted by the immunoglobulins of the present invention. Autoimmune and inflammatory targets include, but are not limited to, C5, CCL5 (I-309), CCL5 (eosinophil chemokine), CCL5 (mcp-4), CCL 5(MIP-1d), CCL5 (HCC-4), CCL5 (TARC), CCL5 (PARC), CCL5, CCL5 (mcp-1), CCL5 (MIP-3a), CCL5 (MIP-2), CCL5 (MPIF-1), CCL5 (MPIF-2/eosinophil chemokine-2), CCL 5(TECK), CCL5, CCL 5(MIP-1 a), CCL5 (MIP-RANb), CCL5 (TES), CCL5 (mcp-3), CCL5 (mcp-2), CXCL5, CXCL5 (IP-10), CXCL5 (ENA/IP-9), CXCL5 (SDF 5), CXCL5, CXCL5 (CXCL 5, CXCL5, CXCL-5 (CXCL-5, 5 (CXCL-5), CXCL-5 (TAC-5), CXCL-5, 5) and 5 (CXCL-5) 5 (CXCL-5, CXCL-5 (TAC-5) 5, CXCL-5, 5 (TAC-5) and CXCL-5 (TAC-5), CCX-5 (TAC-5, 5) and 5 (TAC-5), CCX-5 (TAC-5) and CAC 5, CCL5 (TAC-5) and CAC 5 (TAC-5), IL, IL, CCL (mcp-4), CCR, CCR, CCR, CCR, CCR, CCR, CCR, CCR, CCR 1F, CX3CR, IL8RA, XCR (CCXCR), IFNA, IL, IL17, IL1, IL1, IL1F, IL1F, IL1F, IL1F, IL, IL, IL, IL, LTA, LTB, MIF, SCYE (endothelial monocyte activating cytokine), SPP, TNF, TNFSF, IFNA, IL10RA, IL10RB, IL, IL13RA, IL5RA, IL, IL9, ABCF, BCL, C, C4, CEBPB, CRP, ICEBERG, IL1R, IL1RN, IL8RB, LTB4, TOLLIP, IRAK, IRAK, ABCD, FADK, TNFR, TRAFF, TRAVR, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL-CR, CCL-3 CR, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCL, CCR, CCR, CCR, CCR, CCR, XC, CCR, CX3CL, CX3CR, CXCL, CXCL, CXCL, CXCL, CXCL, GPR, SCYE, SDF, XCL, XCL, XCR, AMH, AMHR, BMPR1, BMPR, C19orf (IL 27), CER, CSF, CSF, CSF, DKFZsp 451J0118, FGF, GFI, IFNA, IFNB, IFNG, IGF, IL1, IL1R, IL1R, IL, IL2RA, IL2RB, IL, IL, IL4, IL, IL5RA, IL, IL6, IL6ST, GF, IL, IL8RA, IL9, IL RA, IL10RB, IL12RB, TNFRSF, TNFRSF, TNFRSF, TNFRSF 12, TNFRSF, TNFRSF, TNFRSF, CX, CXFB, CX, CXFB, CX, CXFB, CX, IFB, CX, IFB, IF, TNFSF11, VEGF, ZFPM2 and RNF110(ZNF 144). To form a bispecific or trispecific antibody of the invention, antibodies can be prepared against any combination of these antigens; that is, each of these antigens may optionally and independently be included or excluded by multispecific antibodies according to the present invention.

Exemplary common targets for autoimmune and inflammatory disorders include, but are not limited to, I L-1 and TNF α, I α 0-6 and TNF α, I α 1-6 and I α 2-1, IgE and I α 3-13, I α 4-1 and I L-13, I L-4 and I L-13, I L-5 and I L-13, I L-9 and I L-13, CD19 and Fc γ RIIb, and CD79 and Fc γ RIIb.

The immunoglobulins of the present invention contemplated to be specific for the target pairs TNF and I-17A, TNF and RANK, TNF and VEGF, TNF and SOST, TNF and DKK, TNF and V3, TNF and NGF, TNF and I0-23 p, TNF and I1-6, TNF and SOST, TNF and I2-6R, TNF and CD-20, IgE and I3-13, I4-13 and I523 p, IgE and I6-4, IgE and I7-9, IgE and I8-9, IgE and I9-13, I0-13 and I1-9, I2-13 and I3-4, I4-13 and I5-9, I6-13 and I7-9, I8-13 and I9-4, I0-13 and I1-23 p, I2-13 and I3-9, I4-6R and VEGF, I6-6R and I17-9, I8-13 and I9-4, I0-13-23 p, I2-13 and I3-9, I4-6R and VEGF, I6-6R and I17A, I1-7-9, I8-4, I1-7-9, I1-23 p, I2-9, I2-1-7, and I1-9, and I1-7, and I1-4, and 5, and I1-9, and 5, and 1-9, and 1-1, and 4, and 1-1.

In one embodiment, such targets include but are not limited to I-13 and I-1 8, as I0-1 is also associated with inflammatory responses in asthma, I1-13 is associated with cytokines and chemokines involved in inflammation, such as I2-13 and I3-9, I4-13 and I5-4, I6-13 and I7-5, I9-13 and I0-25, I1-13 and TARC, I2-13 and MDC, I3-13 and MIF, I4-13 and TGF-; I5-13 and 6 agonists, I7-13 and C825, I9-13 and SPRR2, I-13 and SPRR2, and I0-13 and ADAM 8. immunoglobulins herein may be made to one or more of the targets involved in asthma selected from the group consisting of CSF (CSF) CSF 23, CSF 7-13 (CSF), CXCR 7-13, CXCR 7, CXCR 53, CXCR1, CXCR 53, CXCR < 1 > C > and CXCR < 1 > 72 > C > 53 < 1, CXCR < 1 > and CXCR < 1, CXCR < 1 > 72 > C < 1 > C < 1 > 1 < 1 > C < 1 > C < 1 > C < 1 > C < 1 > 2 < 1 < 11 > C < 1 < 11 > C < 1 > C < 1 > C < 1 > C < 1 > C < 1 > C < 1 > C < 1 > C < 1 > C < 1 > C < 1 > C < 1.

Target pairs involved in Rheumatoid Arthritis (RA) may be co-targeted by the present invention, including but not limited to TNF and I L-18, TNF and I L-12, TNF and I L-23, TNF and I L-1 β, TNF and MIF, TNF and I L-17, and TNF and I L-15.

The antigen that can be targeted by the immunoglobulins described herein for the treatment of systemic lupus erythematosus (S L E) includes, but is not limited to, CD-20, CD-22, CD-19, CD28, CD4, CD80, H L A-DRA, I L010, I L12, I L24, TNFRSF5, TNFRSF6, TNFRSF5, TNFRSF6, B L3R 1, HDAC4, HDAC5, HDAC7A, 9, ICOS L4, IGBP1, MS4A1, SI, S L5A L, CD L, 685NB L, I L610, TNFRSF L, TNFRSF L, AICDK, B L NK 7, GA L-6 ST, HDAC L, L, L DRA7, HDAC L, L I L, L D L, L A L, L D L, L A L, L A L, L A L, L A, L A L, L A, L, and K, L T, L T, and K, or a with the invention can be combined with the antibody with the present in a, or T L T, or T, and the invention can be made according to the present invention, with the present invention, and the present in the invention, or the present invention, and the invention, or the present invention, CD L, or the antibody of the invention, or the CD L, or the invention, or the antibody of the invention, or.

The immunoglobulins herein may be targeted to antigens useful in the treatment of Multiple Sclerosis (MS), including but not limited to I L-12, TWEAK, I L-23, CXC L13, CD40, CD 40L, I L-18, VEGF, V L A-4, TNF, CD45RB, CD200, IFN γ, GM-CSF, FGF, C5, CD52 and CCR2 one embodiment includes co-conjugating anti-I L-12 and TWEAK to treat MS.

One aspect of the present invention relates to an immunoglobulin capable of binding to one or more targets involved in sepsis, in one embodiment binding to two targets selected from the group consisting of TNF, I L-1, MIF, I L-6, I L-8, I L-18, I L-12, I L3-23, Fas L PS, Toll-like receptor, T L R-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4, I L-10, I L-1B, NF kappa B1, PROC, TNFRFIA, CSF3, CCR3, I3 IRN, MIF, NF kappa B3, PTAFR, T3R 3, T3R 3, 685GPR 4, HMOX 3, NF kappa factor kappa B3, NF 3, MID-NI-3, and optionally a bispecific antibody may be prepared according to the present invention, or in order to form a bispecific antibody against each of these antigens, optionally in a combination, or in order to form a bispecific antibody against the midkine.

In some cases, the immunoglobulins herein may be directed against antigens used to treat infectious diseases.

Antigen binding domains

As will be appreciated by those skilled in the art, there are two basic types of antigen binding domains, those that resemble antibody antigen binding domains (e.g., comprise a set of 6 CDRs) and those that may be ligands or receptors, e.g., those that do not require the use of CDRs to bind a target.

Modified antibodies

For example, the molecule may be stabilized by incorporation of disulfide bonds linking the VH and V L domains (Reiter et al, 1996, Nature Biotech.14:1239-1245, incorporated by reference in its entirety).

Covalent modification of antibodies is included within the scope of the invention and is typically, but not always, done post-translationally. For example, several types of covalent modifications of antibodies are introduced into the molecule by reacting specific amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or N-or C-terminal residues.

Cysteinyl residues are most commonly reacted with α -haloacetates (and the corresponding amines, such as chloroacetic acid or chloroacetamide) to give carboxymethyl or carboxyamidomethyl derivatives cysteinyl residues can also be derivatized by reaction with bromotrifluoroacetone, α -bromo- β - (5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercurybenzoate, 2-chloromercury-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1, 3-diazole, or the like.

In addition, modifications at the cysteine residues are particularly useful in antibody-drug conjugate (ADC) applications, as further described below. In some embodiments, the constant region of the antibody may be engineered to contain one or more cysteines (which are "thiol-reactive") in order to allow more specific and controllable placement of the drug moiety. See, for example, U.S. patent No. 7,521,541, which is incorporated by reference herein in its entirety.

Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl methyl bromide may also be used; the reaction is preferably carried out in 0.1M sodium cacodylate at pH 6.0.

Other suitable reagents for derivatizing the α -amino-containing residue include imidoesters such as methyl-must-imido-methyl ester, pyridoxal phosphate, pyridoxal, boron-hydride chloride, trinitrobenzenesulfonic acid, O-methylisourea, 2, 4-pentanedione, and transaminatively catalyzed reactions with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among which are benzoylformaldehyde, 2, 3-butanedione, 1, 2-cyclohexanedione and ninhydrin. Derivatization of arginine residues requires that the reaction be performed under alkaline conditions because the guanidine functional group has a high pKa. In addition, these reagents can react with lysine as well as arginine-amino groups.

Specific modification of tyrosyl residues can be performed, with particular interest in introducing spectroscopic tags into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125I or 131I to prepare labeled proteins for radioimmunoassay, the chloramine T method described above being suitable.

Pendant carboxyl groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimide (R ' -N ═ C ═ N-R '), where R and R ' are optionally different alkyl groups, such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4, 4-dimethylpentyl) carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional reagents can be used to crosslink antibodies to water-insoluble support matrices or surfaces for use in a number of methods, other than those described below. Commonly used crosslinking agents include, for example, 1, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imides, including disuccinimidyl esters such as 3, 3' -dithiobis (succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1, 8-octane. Derivatizing agents such as methyl-3- [ (p-azidophenyl) dithio ] propiminate produce photo-activated intermediates that are capable of forming crosslinks in the presence of light. Alternatively, water-insoluble reaction substrates such as those described in U.S. patent nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 (all incorporated by reference in their entirety) were used for protein immobilization.

Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Any form of these residues is within the scope of the present invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α -amino group of the lysine, arginine and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp.79-86[1983], incorporated by reference in its entirety), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl groups.

In addition, labels (including fluorescent, enzymatic, magnetic, radioactive, etc.) can be added to the antibodies (as well as other compositions of the invention), as will be appreciated by those skilled in the art.

Glycosylation

Another type of covalent modification is glycosylation changes. In another embodiment, the antibodies disclosed herein may be modified to comprise one or more engineered glycoforms. An "engineered glycoform" as used herein means a saccharide composition covalently attached to an antibody, wherein the saccharide composition is chemically distinct from that of the parent antibody. The engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or reducing effector function. A preferred form of engineered glycoform is afucosylation, which has been shown to be associated with increased ADCC function, presumably through tighter binding to the Fc γ RIIIa receptor. In this context, "afucosylation" means that the majority of antibodies produced in a host cell are substantially free of fucose, e.g., 90-95-98% of the antibodies produced do not have an equivalent amount of fucose as a component of the carbohydrate portion of the antibody (typically attached at N297 of the Fc region). Functionally defined, afucosylated (afucosylated) antibodies typically show an affinity for Fc γ RIIIa receptors of at least 50% or higher.

The engineered glycoforms can be produced by a variety of methods known in the art: (

Et al, 1999, NatBiotechnol 17: 176-180; davies et al, 2001, Biotechnol Bioeng 74: 288-294; shield et al, 2002, J Biol Chem 277: 26733-; shinkawa et al, 2003, J Biol Chem 278: 3466-; US 6,602,684; USSN 10/277,370; USSN 10/113,929; PCT WO 00/61739a 1; PCT WO01/29246A 1; PCT WO 02/31140a 1; PCT WO 02/30954a1, all incorporated by reference in their entirety; (

technology[Biowa,Inc.,Princeton,NJ]；

glycosylation engineering technology [ Glycart Biotechnology AG, Z ü rich, Switzerland]) Many of these techniques are based on controlling the level of fucosylated and/or bisected oligosaccharides covalently attached to an Fc region, e.g., by expressing IgG in various organisms or cell lines that are engineered or otherwise (e.g., L ec-13 CHO cells orRat hybridoma YB2/0 cell, by regulating enzymes involved in glycosylation pathway (e.g. FUT8[ α 1, 6-fucosyltransferase)]And/or β 1-4-N-acetylglucosaminyltransferase III [ GnTIII ]]) Or by modifying one or more of the sugars after the IgG has been expressed. For example, the "glycoengineered antibodies" or "SEA technology" of Seattle Genetics works by adding modified sugars that inhibit fucosylation during production; see, e.g., 20090317869, incorporated herein by reference in its entirety. Engineered glycoforms typically refer to different saccharides or oligosaccharides; the antibody may thus comprise an engineered glycoform.

Alternatively, an engineered glycoform may refer to an IgG variant comprising different saccharides or oligosaccharides. As is known in the art, the glycosylation pattern can depend on the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, discussed below), or both the host cell or organism producing the protein. Specific expression systems are discussed below.

Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of a sugar moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the sugar moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide forms a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid (most commonly serine or threonine), although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antibody is conveniently achieved by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be obtained by addition to or replacement by one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). For convenience, the antibody amino acid sequence is preferably altered by changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.

Another means of increasing the number of sugar moieties on an antibody is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require the production of proteins in host cells that have the glycosylation capability to carry out N-or O-linked glycosylation. Depending on the coupling mode used, one or more sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 and Aplin and Wriston,1981, CRC crit. rev. biochem., pp.259-306, both of which are incorporated by reference in their entirety.

Removal of the carbohydrate moiety present on the starting antibody (e.g., post-translational) can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposing the protein to the compound triflic acid, or an equivalent compound. This treatment results in the cleavage of most or all of the sugars except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al, 1987, Arch.Biochem.Biophys.259:52 and Edge et al, 1981, anal.Biochem.118:131, both of which are incorporated by reference in their entirety. Enzymatic cleavage of the sugar moiety on a polypeptide can be achieved by using a variety of endo-and exo-glycosidases, as described by Thotakura et al, 1987, meth.enzymol.138:350, which is incorporated herein by reference in its entirety. Glycosylation at potential glycosylation sites can be prevented by the use of the compound tunicamycin, as described by Duskin et al, 1982, J.biol.chem.257:3105, incorporated herein by reference in its entirety. Tunicamycin blocks the formation of protein-N-glycosidic bonds.

Another type of covalent modification of an antibody includes linking the antibody to a variety of non-proteinaceous polymers, including, but not limited to, a variety of polyols such as polyethylene glycol, polypropylene glycol, or polyalkylene oxide, in the manner set forth in the following literature: for example, 2005-2006PEG Catalog from Nektar Therapeutics (available at the Nektar website), U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, all incorporated by reference in their entirety. In addition, as is known in the art, amino acid substitutions can be made in various positions within the antibody to facilitate the addition of polymers such as PEG. See, for example, U.S. publication No. 2005/0114037a1, incorporated by reference in its entirety.

Other Fc variants for other functionalities

In addition to pI amino acid variants, a number of useful Fc amino acid modifications can be made for a variety of reasons, including, but not limited to, altering binding to one or more Fc γ R receptors, altering binding to FcRn receptors, and the like.

Thus, the proteins of the invention may comprise amino acid modifications, including heterodimerization variants as outlined herein, including pI variants and steric variants. Each set of variants may be independently and optionally included or excluded by any particular heterodimeric protein.

Fcyr variants

Thus, a number of useful Fc substitutions can be made to alter binding to one or more fcyr receptors, substitutions resulting in increased binding as well as decreased binding can be useful, for example, it is known that increased binding to Fc RIIIa generally results in increased ADCC (antibody-dependent cell-mediated cytotoxicity; cell-mediated reactions in which non-specific cytotoxic cells expressing fcyr recognize bound antibody on target cells, subsequently resulting in lysis of target cells.) similarly, reduced binding to Fc γ RIIb (inhibitory receptor) can also be beneficial in some cases.

In addition, there are other Fc substitutions that can be used to increase binding to the FcRn receptor and increase serum half-life, as specifically disclosed in USSN 12/341,769, which is incorporated herein by reference in its entirety, including but not limited to 434S,434A, 428L, 308F,259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L.

Joint

The invention optionally provides linkers as desired, e.g., upon addition of additional antigen binding sites, as depicted, for example, in fig. 2, wherein the "other end" of the molecule contains additional antigen binding components. In addition, as outlined below, linkers are also optionally used in Antibody Drug Conjugate (ADC) systems. When used to join components of a central mAb-Fv construct, a linker is typically a polypeptide comprising two or more amino acid residues joined by peptide bonds and is used to join one or more components of the invention. Such linker polypeptides are well known in the art (see, e.g., Holliger, P., et al (1993) Proc. Natl. Acad. Sci. USA 90: 6444-. In some embodiments described herein, it is found that a variety of linkers can be used. As will be appreciated by those skilled in the art, there are at least three different types of linkers for use in the present invention.

"linker" is also referred to herein as "linker sequence", "spacer", "tether sequence" or grammatical equivalents thereof. Homo-or hetero-bifunctional linkers are well known (see, 1994Pierce Chemical Company catalog, technical section cross-linkers, technical section on crosslinkers, page 155-200, incorporated by reference in its entirety). Many strategies can be used to covalently link the molecules together. These strategies include, but are not limited to, polypeptide linkage between the N-and C-termini of proteins or protein domains, linkage via disulfide bonds, and linkage via chemical crosslinking agents. In one aspect of this embodiment, the linker is a peptide bond, produced by recombinant techniques or peptide synthesis. The linker peptide may comprise predominantly the following amino acid residues: gly, Ser, Ala or Thr. The linker peptide should be of sufficient length to link the two molecules such that they assume the correct configuration relative to each other such that they retain the desired activity. In one embodiment, the linker is about 1-50 amino acids in length, preferably about 1-30 amino acids in length. In one embodiment, linkers of 1-20 amino acids in length may be used. Useful linkers include glycine-serine polymers including, for example, (GS) n, (GSGGS) n, (GGGGS) n, and (GGGS) n, where n is an integer of at least 1, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, a variety of non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyalkylene oxide, or copolymers of polyethylene glycol and polypropylene glycol may be used as linkers.

Other linking sequences may include any sequence of any length of all residues of the C L/CH 1 domain but not the C L/CH 1 domain, e.g., the first 5-12 amino acid residues of the C L/CH 1 domain.

Antibody-drug conjugates

In some embodiments, the multispecific antibodies of the present invention are conjugated to a drug to form antibody-drug conjugates (ADCs). Typically, ADCs are used in tumor applications where the use of antibody-drug conjugates for local delivery of cytotoxic or cytostatic agents allows for the targeted delivery of drug moieties to tumors, which may allow for higher efficacy, lower toxicity, etc. For an overview of this technology, see Ducry et al, Bioconjugate chem.,21:5-13(2010), Carter et al, Cancer J.14(3):154(2008) and Senter, Current opinion, chem.biol.13:235-244(2009), all of which are incorporated herein by reference in their entirety.

In general, conjugation is accomplished by covalent attachment to an antibody, as described further below, and typically relies on a linker, often a peptide bond (which may be designed to be sensitive or insensitive to cleavage by proteases at target sites, as described below). additionally, as described above, attachment of linker-drug units (L U-D) may be accomplished by attachment to cysteines within the antibody.

The invention thus provides multispecific antibodies conjugated to a drug. As described below, the drug of the ADC can be any number of agents, including but not limited to providing a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate). In other embodiments, the invention also provides methods of using the ADCs.

Drugs for use in the present invention include cytotoxic drugs, particularly those for use in the treatment of cancer. Such agents include, in general, DNA damaging agents, antimetabolites, natural products and the like. Exemplary classes of cytotoxic agents include enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNA cleaving agents, topoisomerase inhibitors, anthracycline family drugs, vinca drugs, mitomycins, bleomycin, cytotoxic nucleosides, pteridine family drugs, diynes (diynenes), podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and paclitaxel.

Members of these classes include, for example, methotrexate dichloride, 5-fluorouracil, 6-mercaptopurine, cytarabine, melphalan, lomonone, leurosidine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tersulosin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including paclitaxel, taxotere retinoic acid, butyric acid, N8-acetylspermine, camptothecin, calicheamicin, estomycin, ene-diynes, duocarmycin A (duocarmycin A), duocarmycin SA, calicheamicin, camptothecin, maytansinoids compounds (including DM1), monomethyl auristatin E (monomethylauristatin E), monomethyl aurine E (MMAE), monomethyl auristatin F (MMAE), and maytansinoids (DM4) and their analogs.

Toxins may be used as antibody-toxin conjugates and include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst.92(19):1573 1581; Mandler et al (2000) Bioorganic & Med. chem. L ets 10: 1025-.

Conjugates of multispecific antibodies and one or more small molecule toxins, such as maytansinoids, dolastatins, auristatins, trichothecenes, calicheamicins, and CC1065, as well as derivatives of these toxins that have toxin activity, are contemplated.

Maytansinoids

Maytansinoid compounds suitable for use as the drug moiety of maytansinoids are well known in the art and may be isolated from natural sources according to known methods, prepared using genetic engineering techniques (see Yu et al (2002) PNAS 99: 7968-. As described below, the drug may be modified by the addition of a functionally active group such as a thiol group or an amine group for conjugation to the antibody.

Exemplary maytansinoid drug moieties include those having modified aromatic rings, such as C-19-dechlorination (U.S. Pat. No. 4,256,746) (prepared by lithium aluminum hydride reduction of ansamitocin P2), C-20-hydroxy (or C-20-demethyl) +/-C-19-dechlorination (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or by dechlorination using L AH), and C-20-demethoxy, C-20-acyloxy (- -OCOR), +/-dechlorination (U.S. Pat. No. 4,294,757) (prepared by acylation using an acyl chloride) and those having modifications at other positions.

Exemplary maytansinoid drug moieties also include those having modifications such as C-9-SH (U.S. Pat. No. 4,424,219) (prepared by reaction of maytansinol with H2S or P2S 5), C-14-alkoxymethyl (demethoxy/CH 2OR) (U.S. Pat. No. 4,331,598), C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2Oac) (U.S. Pat. No. 4,450,254) (prepared by Nocardia), C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by Streptomyces conversion of maytansinol), C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudiflora), C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by Streptomyces demethylation of maytansinol), and 4, 5-deoxy (U.S. Pat. No. 4,371,533) (prepared by reduction of maytansinol by titanium trichloride/L).

Of particular use are DM1 (disclosed in U.S. patent No. 5,208,020, incorporated by reference) and DM4 (disclosed in U.S. patent No. 7,276,497, incorporated by reference). See also 5,416,064, WO/01/24763,7,303,749,7,601,354, USSN 12/631,508, WO02/098883,6,441,163,7,368,565, WO02/16368 and WO04/1033272 for a number of other maytansinoid derivatives and methods, all of which are specifically incorporated by reference in their entirety.

ADCs containing maytansinoids, methods for their preparation and their therapeutic use are disclosed in, for example, U.S. Pat. Nos. 5,208,020, 5,416,064, 6,441,163 and European patent EP 0425235B 1, the disclosures of which are specifically incorporated herein by reference L iu et al, Proc. Natl. Acad. Sci. USA 93:8618-8623(1996) describe ADCs containing maytansinoids called DM1 linked to monoclonal antibody C242 directed to human colon cancer.

Chari et al, Cancer Research 52:127- & 131(1992) describe ADCs in which maytansinoids are conjugated via disulfide linkers to the murine antibody A7, which binds to an antigen on a human colon carcinoma cell line, or to another murine monoclonal antibody TA.1, which binds to the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3x105HER-2 surface antigen per cell. The degree of cytotoxicity achieved by the drug conjugates is similar to that of free maytansinoid drugs, and can be increased by increasing the number of maytansinoid molecules per antibody molecule. The a 7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

Auristatin and dolastatin

In some embodiments, the ADC comprises a multispecific antibody conjugated to dolastatin or dolastatin peptide analogs and derivatives, auristatin (U.S. Pat. nos. 5,635,483; 5,780,588). Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother.45(12):3580-3584) and to have anti-cancer activity (U.S. Pat. No. 5,663,149) and anti-fungal activity (Pettit et al (1998) Antimicrob. Agents Chemother.42: 2961-2965). Dolastatin or auristatin drug moieties can be attached to an antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminally attached monomethyl auristatin drug moieties DE and DF, as disclosed in "Senter et al, Proceedings of the American Association for cancer research, Volume 45, Abstract Number 623, presented Mar.28,2004 and in U.S. patent publication No. 2005/0238648, the disclosure of which is specifically incorporated by reference in its entirety.

One exemplary auristatin embodiment is MMAE (see U.S. patent No. 6,884,869, which is specifically incorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF (see US 2005/0238649,5,767,237, and 6,124,431, specifically incorporated by reference in its entirety).

Other exemplary embodiments comprising MMAE or MMAF and various linker components (further described herein) have the following structures and abbreviations (wherein Ab means antibody and p is 1 to about 8):

peptide-based Drug moieties can typically be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments this peptide bond can be prepared, for example, according to liquid phase Synthesis methods well known in The art of peptide chemistry (see E.Schroder and K. L ubke, "The Peptides", volume 1, pp 76-136,1965, Academic Press). Amitastatin/dolastatin Drug moieties can be prepared according to The following methods, U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J.am. chem. Soc.111: 5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13: 243-277; Pettit, G.R., et al Synthesis,1996,725; Pettit et al (1996) J.chem. c.15. peptide fragments: 789: 779; Natrons 773: 778; Natron. A. 773; Natrons 778).

Calicheamicin

In other embodiments, the ADC comprises an antibody of the invention conjugated to one or more calicheamicin molecules Mylotarg is the first commercially available ADC drug and utilizes calicheamicin γ 1 as the payload (see U.S. Pat. No. 4,970,198, incorporated by reference in its entirety.) other calicheamicin derivatives are described in U.S. Pat. Nos. 5,264,586,5,384,412,5,550,246,5,739,116,5,773,001,5,767,285 and 5,877,296, all of which are specifically incorporated by reference for the preparation of conjugates of the calicheamicin family, QFfamily antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. for the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586,5,116, 5,767,285,5,770,770,701, 5,779,710,5,773,001, 5,877,296, 5,1998, 5,1998,730,586, 5,862,3358,862, the entire strain belonging to the calicheamicin the Canamicin family, and the aforementioned anti-tumour agent (R A), which is not limited to the aforementioned anti-S. AG A antibody, S. AG 2, incorporated by the aforementioned Adenomics A, and the aforementioned anti-S. A antibody, which mediates the effects of the aforementioned anti-A receptor agonist, and the effects of the aforementioned anti-A receptor analogs thereof, and the aforementioned anti-interferon analogs thereof, such as a receptor molecules (S. 2) and the anti-S. Pat. 2, which are greatly enhance the aforementioned anti-influenza A receptor molecules.

Duocarmycin

CC-1065 (see 4,169,888, incorporated by reference) and duocarmycin are members of the family of antitumor antibiotics used in ADCs. These antibiotics appear to act by sequence-selectively alkylating DNA at N3 of adenosine in the minor groove, which initiates a cascade of events leading to apoptosis.

Important members of duocarmycins include duocarmycin a (U.S. Pat. No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat. No. 5,101,038, incorporated by reference), and as described in U.S. Pat. nos. 7,517,903,7,691,962,5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780; 5,101,038; 5,084,468,5,475,092,5,585,499,5,846,545, WO2007/089149, WO2009/017394a1,5,703,080,6,989,452,7,087,600,7,129,261,7,498,302, and 7,507,420, all of which are specifically incorporated by reference.

Other cytotoxic agents

Other anti-tumor agents that may be conjugated to the antibodies of the invention include BCNU, streptozotocin, vincristine and 5-fluorouracil, a family of agents collectively referred to as the LL-E33288 complex described in U.S. patent nos. 5,053,394,5,770,710, and esperamicin (U.S. patent No. 5,877,296).

Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α -sarcin, Aleurites fordii protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitors, curcin, crotin, Fumaria ferox (sapaonaria officinalis) inhibitors, gelonin, mitogellin (mitogellin), restrictocin, phenomycin, enomycin, and trichothecene see, for example, WO 93/21232, 28.10.1993.

The invention further contemplates ADCs formed between the antibody and the compound having nucleolytic activity (e.g., ribonucleases or DNA endonucleases such as deoxyribonucleases; DNases).

A variety of radioisotopes are available for the production of radio-conjugated antibodies.examples include radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and L u.

Radioactive or other labels may be incorporated into the conjugate in known ways. For example, peptides can be biosynthesized or can be synthesized by chemical amino acid synthesis using suitable amino acid precursors including, for example, fluorine-19 in place of hydrogen. Labels such as Tc99m or I123, Re186, Re188 and In111 may be attached via a cysteine residue In the peptide. Yttrium-90 may be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) biochem. Biophys. Res. Commun.80:49-57) may be used to incorporate iodine-123. Other methods are described in detail in "Monoclonal antibodies Immunoscintigraphy (Monoclonal antibodies in Immunoscintigraphy)" (Chatal, CRC Press 1989).

For compositions comprising multiple antibodies, the drug load is represented by p, the average number of drug molecules per antibody the drug load can be 1-20 drug (D)/antibody the average number of drugs per antibody in the preparation of the conjugation reaction can be characterized by conventional means such as mass spectrometry, E L ISA determination, and HP L C.

In some cases, isolation, purification, and characterization of homogeneous antibody-drug-conjugates (where p is a particular value from antibody-drug-conjugates with other drug loadings) can be achieved by means such as reverse phase HP L C or electrophoresis.

The production of the antibody-drug conjugate compound can be achieved by any technique known to the skilled person. Briefly, an antibody-drug conjugate compound may include a multispecific antibody as an antibody unit, a drug, and optionally a linker connecting the drug and a binding agent.

Many different reactions can be utilized for covalent attachment of drugs and/or linkers to the binding agent. This can be achieved by reaction of amino acid residues of the binding agent (e.g., antibody molecule), including amine groups of lysine, free carboxylic acid groups of glutamic acid and aspartic acid, thiol groups of cysteine, and portions of aromatic amino acids. A common non-specific method of covalent attachment is a carbodiimide reaction to attach the carboxyl (or amino) group of a compound to the amino (or carboxyl) group of an antibody. Alternatively, bifunctional agents such as dialdehydes or imidates have been used to link the amino group of a compound to an amino group of an antibody molecule.

Also useful for the attachment of the drug to the binding agent is a schiff base reaction. The method comprises periodic acid oxidation of a drug containing a diol or hydroxyl group, thereby forming an aldehyde, which is then reacted with a binding agent. The linkage occurs through the formation of a schiff base with the amino group of the binding agent. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the skilled person and are within the scope of the invention.

In some embodiments, the intermediate that is a precursor to the linker is reacted with the drug under appropriate conditions. In other embodiments, reactive groups are used on the drug and/or the intermediate. The reaction product between the drug and the intermediate, or derivatized drug, is then reacted with the multispecific antibodies of the present invention under appropriate conditions.

It will be appreciated that chemical modifications may also be made to the desired compound in order to facilitate the reaction of the compound for the purpose of preparing the conjugates of the invention. For example, a functional group such as an amine, hydroxyl, or sulfhydryl group, may be attached to a drug at a location that has minimal or acceptable effect on the activity or other properties of the drug.

ADC connector unit

Typically, the antibody-drug conjugate compound comprises a linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular or extracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in a suitable environment. For example, solid tumors that secrete certain proteases can serve as targets for cleavable linkers; in other embodiments, it is an intracellular protease that is utilized. In still other embodiments, the linker unit is non-cleavable, and for example, the drug is released by antibody degradation within the lysosome.

In some embodiments, the linker can be cleaved by a cleaving agent present in the intracellular environment (e.g., lysosome or endosome or endocellular). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease (including, but not limited to, lysosomal or endosomal proteases). In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long or longer.

Lytic agents can include, but are not limited to, cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in release of the active drug in target cells (see, e.g., Dubowchik and Walker,1999, pharm. therapeutics 83: 67-123.) peptidyl linkers cleavable by enzymes present in cells expressing CD38 can be used, e.g., peptidyl linkers cleavable by thiol-dependent proteases, cathepsin-B (which is highly expressed in cancerous tissue) (e.g., Phe-L eu or Gly-Phe-L eu-Gly linker (SEQ ID NO: X)). other examples of such linkers are described, for example, in U.S. patent No. 6,214,345, which is incorporated by reference in its entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-L ys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a Val-Cit linker).

In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a particular pH value. Typically, the pH-sensitive linker is hydrolysable under acidic conditions. For example, acid-labile linkers that are hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) may be used. (see, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker,1999, pharm. therapeutics 83: 67-123; Neville et al, 1989, biol. chem.264:14653-14661.) such linkers are relatively stable under neutral pH conditions, such as those in blood, but are unstable below pH 5.5 or 5.0, which is approximately the pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, for example, a thioether linked to a therapeutic agent via an acylhydrazone bond (see, e.g., U.S. patent No. 5,622,929).

In still other embodiments, the linker is cleavable under reducing conditions (e.g., disulfide linker). A number of disulfide linkers are known In the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate), SPDB (N-succinimidyl-3- (2-pyridyldithio) butyrate) and SMPT (N-succinimidyl-oxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene) -, SPDB and SMPT (see, e.g., Thorpe et al, 1987, Cancer Res.47: 5924;. Wawrzynczak et al, In Immunoconjunctes: Antibodies Conjugates In radionuclides, of Cancer (C.W.Vogel ed., Oxford.38U.S. Pat. No. 1987. Pre 4,880,935.)

In other embodiments, the linker is a malonate linker (Johnson et al, 1995, anticancer Res.15:1387-93), a maleimidobenzoyl linker (L au et al, 1995, Bioorg-Med-chem.3(10):1299-1304) or a 3' -N-amide analog (L au et al, 1995, Bioorg-Med-chem.3(10): 1305-12).

In still other embodiments, the linker unit is non-cleavable and the drug is released by antibody degradation. (see U.S. publication No. 2005/0238649, which is incorporated herein by reference in its entirety and for all purposes).

In many embodiments, the linker is self-immolative. As used herein, the term "self-immolative spacer" refers to a bifunctional chemical moiety capable of covalently linking two spaced chemical moieties together into a stable three-body molecule. If its bond to the first moiety is cleaved, it spontaneously separates from the second chemical moiety. See, for example, WO 2007059404a2, WO06110476a2, WO05112919a2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431, WO04/043493, and WO02/083180, which are directed to drug-cleavable substrate conjugates in which the drug and cleavable substrate are optionally linked by a self-shedding linker, and all patent applications are specifically incorporated by reference.

The linker is often substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" in the context of a linker means that no more than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of the linker in a sample of an antibody-drug conjugate compound is cleaved when the antibody-drug conjugate compound is present in the extracellular environment (e.g., in plasma).

It can be determined whether the linker is substantially insensitive to the extracellular environment, for example, by incubating the plasma with the antibody-drug conjugate compound for a predetermined period of time (e.g., 2,4,8,16, or 24 hours), and then quantifying the amount of free drug present in the plasma.

In other embodiments that are not mutually exclusive, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic agent portion of an antibody-drug conjugate compound as described herein). In still other embodiments, the linker promotes cellular internalization when conjugated to both an auristatin compound and a multispecific antibody of the present invention.

A variety of exemplary linkers that can be used in the compositions and methods of the invention are described in WO 2004-010957, U.S. publication No. 2006/0074008, U.S. publication No. 20050238649, and U.S. publication No. 2006/0024317 (each of which is incorporated by reference in its entirety and for all purposes).

Drug loading

The drug loading ("p") can be 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more moieties (D) per antibody, although the average number is often a fraction or decimal.generally, drug loadings of 1-4 are often useful, and 1-2 are also useful.

The number distribution of ADCs in terms of p can also be determined. In some cases, separation, purification, and characterization of homogeneous ADCs (where p is a particular value from ADCs with other drug loadings) may be achieved by means such as electrophoresis.

For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, in the case of attachment to a cysteine thiol, as in the exemplary embodiments above, the antibody may have only one or a few cysteine thiol groups, or may have only one or a few sufficiently reactive thiol groups through which a linker may be attached. In certain embodiments, higher drug loadings, e.g., p >5, can result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the drug loading of the ADCs of the present invention ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody may be less than 8, and may be from about 2 to about 5. See US 2005-0238649 a1 (incorporated herein by reference in its entirety).

In certain embodiments, less than the theoretical maximum of drug moieties are conjugated to the antibody during the conjugation reaction. The antibody may comprise, for example, lysine residues that are not reactive with the drug-linker intermediate or linker reagent, as discussed below. Typically, antibodies do not contain many free and reactive cysteine thiol groups, which may be attached to a drug moiety; in fact most cysteine thiol residues in antibodies are present as disulfide bonds. In certain embodiments, the antibody can be reduced with a reducing agent such as Dithiothreitol (DTT) or Tricarbonylethylphosphine (TCEP) under partially or fully reducing conditions to produce reactive cysteine thiol groups. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

The loading of the ADC (drug/antibody ratio) can be controlled in different ways, for example, by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, (iii) limiting the reduction conditions or moieties to cysteine thiol modification, (iv) engineering the amino acid sequence of the antibody by recombinant techniques such that the number and position of cysteine residues are altered to control the number and/or position of linker-drug linkages (such as thioMab or thioFab prepared as disclosed herein and in WO2006/034488 (incorporated herein by reference in its entirety)).

It is understood that in the case where more than one nucleophilic group reacts with a drug-linker intermediate or linking reagent and then with a drug moiety reagent, then the resulting product is a mixture of ADC compounds in which there are one or more drug moieties attached to the antibody.

In some embodiments, a homogeneous ADC having a single loading value can be separated from the conjugate mixture by electrophoresis or chromatography.

Method for determining the cytotoxic effects of ADC

Methods for determining whether a drug or antibody-drug conjugate exerts a cytostatic and/or cytotoxic effect on a cell are known. In general, the cytotoxic or cytostatic activity of an antibody drug conjugate can be measured by: exposing mammalian cells expressing the target protein of the antibody drug conjugate to a cell culture medium; culturing the cells for a period of about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays can be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis by antibody drug conjugates (caspase activation).

To determine whether an antibody drug conjugate exerts a cytostatic effect, a thymidine incorporation assay may be used. For example, cancer cells expressing a target antigen can be cultured at a density of 5,000 cells/well of 96 wells plated for a period of 72 hours and exposed to 0.5 μ Ci of 3H-thymidine during the last 8 hours of the 72 hour period. The incorporation of 3H-thymidine in cells of the culture was measured in the presence and absence of antibody drug conjugates.

For determining cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by an increase in permeability of the plasma membrane; cell swelling and plasma membrane disruption. Apoptosis is typically mediated by plasma membrane blebbing, cytoplasmic concentration, and activation of endogenous endonucleases. An assay for any of these effects on cancer cells indicates that the antibody drug conjugate is useful in the treatment of cancer.

Cell viability can be measured by measuring the uptake of dyes in cells, such as neutral red, trypan blue or A L AMAR^TMBlue (see, e.g., Page et al, 1993, Intl.J. Oncology 3: 473-. In such assays, cells are incubated in a medium containing a dye, the cells are washed, and the remaining dye, which reflects cellular uptake of the dye, is measured spectrophotometrically. Cytotoxicity can also be measured using the protein-binding dye sulforhodamine b (srb) (Skehan et al, 1990, j. natl. cancer inst.82: 1107-12).

Alternatively, tetrazolium salts, such as MTT, are used in quantitative colorimetric assays for survival and proliferation of mammals by detecting live cells rather than dead cells (see, e.g., Mosmann,1983, J.Immunol. methods 65: 55-63).

Examples of such assays, including TUNE L (which detects incorporation of labeled nucleotides into fragmented DNA) and assays based on the E L ISA, are described in Biochemica,1999, No.2, pp.34-37(Roche molecular biochemicals).

Apoptosis can also be determined by measuring morphological changes in cells. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring uptake of certain dyes (e.g., fluorescent dyes such as, for example, acridine orange or ethidium bromide). One method of measuring the number of apoptotic cells has been described by Duke and Cohen, Current Protocols in immunology (edited by Coligan et al, 1992, pp.3.17.1-3.17.16). Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide) and observed for chromatin condensation and margination along the internal nuclear membrane of the cells. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing and cell shrinkage.

The presence of apoptotic cells can be measured in both the adherent and "floating" compartments of the culture. For example, both compartments can be collected by removing the supernatant, trypsinizing adherent cells, combining the preparations after a centrifugal washing step (e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., by measuring DNA fragmentation). (see, e.g., Piazza et al, 1995, Cancer Research 55: 3110-16).

In vivo, the effect of a therapeutic composition of a multispecific antibody of the present invention may be evaluated in a suitable animal model. For example, a xenogeneic cancer model can be used in which cancer explants or passaged xenogeneic tissue are introduced into immunodeficient animals, such as nude or SCID mice (Klein et al, 1997, Nature Medicine 3: 402-408). Efficacy can be measured using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

Therapeutic compositions used in practicing the foregoing methods may be formulated as pharmaceutical compositions comprising a carrier suitable for use in the desired delivery method. Suitable carriers include any substance that retains the anti-tumor function of the therapeutic composition when combined with the therapeutic composition and that does not normally react with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such asPhosphate buffered saline, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16^thEdition,A.Osal.,Ed.,1980)。

Antibody compositions for in vivo administration

By mixing the antibody with the desired degree of purity and optionally pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16)^thedition,Osol,A.Ed.[1980]) The preparation of the antibody for use according to the present invention in the form of a lyophilized preparation or an aqueous solution is prepared for preservation by mixing. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexa hydroxy quaternary ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN^TM,PLURONICS^TMOr polyethylene glycol (PEG).

The formulations herein may also contain more than one active compound as required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to provide antibodies with other specificities. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine, growth inhibitory agent and/or small molecule antagonist. Such molecules are suitably present in the combination in an amount effective for the desired purpose.

The active ingredient may also be loaded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such a technique is disclosed in Remington's Pharmaceutical Sciences 16^thedition,Osol,A.Ed.(1980)。

The formulation to be used for in vivo administration should be sterile, or nearly sterile. This is readily accomplished by filtration through sterile filtration membranes.

Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules examples of sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ -ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as L UPRON DEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time.

When the encapsulated antibodies are left in the body for a long time, they may denature or aggregate due to exposure to moisture at 37 ℃, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization, depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S — S bond formation through thiol-disulfide interchange, stabilization can be achieved by modifying the thiol residue, lyophilizing from acidic solutions, controlling the water content, using appropriate additives and developing specific polymer matrix compositions.

Administration mode

The antibodies and chemotherapeutic agents of the invention are administered to a subject according to known methods, such as by intramuscular, intraperitoneal, intracerebro-spinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes, as a bolus injection or by intravenous administration as a continuous infusion over a period of time. Intravenous or subcutaneous administration of the antibody is preferred.

Treatment modalities

In the methods of the invention, therapy is used to provide a positive therapeutic response with respect to the disease or condition. By "positive therapeutic response" is meant an improvement in the disease or condition, and/or an improvement in the symptoms associated with the disease or condition. For example, a positive therapeutic response would refer to one or more of the following improvements in the disease: (1) a reduction in the number of tumor cells; (2) increased tumor cell death; (3) inhibition of tumor cell survival; (5) inhibition (i.e., a degree of retardation, preferably cessation) of tumor growth; (6) increased patient survival; and (7) some alleviation of one or more symptoms associated with the disease or condition.

A positive therapeutic response in any given disease or condition can be determined by standardized response criteria specific to that disease or condition. Tumor response changes in tumor morphology (i.e., total tumor burden, tumor size, etc.) can be assessed using screening techniques such as Magnetic Resonance Imaging (MRI) scans, x-ray imaging, Computed Tomography (CT) scans, bone scan imaging, endoscopic and tumor biopsy sampling including Bone Marrow Aspiration (BMA) and circulating tumor cell counts.

In addition to these positive therapeutic responses, subjects undergoing therapy may experience beneficial effects of disease-related symptom improvement.

Thus, for B cell tumors, the subject may experience a reduction in so-called B symptoms, i.e. night sweats, fever, weight loss and/or urticaria. For precancerous lesions, therapy with multispecific therapeutic agents may block the onset of and/or extend the time before onset of a relevant malignant disease, e.g., multiple myeloma in subjects with undefined Monoclonal Gammopathy (MGUS).

Improvement in disease can be characterized as a complete response. By "complete response" is meant the absence of a clinically detectable disease, with any prior abnormal radiographic studies, normalization of bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal proteins in the case of myeloma.

Such a response may last for at least 4-8 weeks, or sometimes 6-8 weeks, following treatment according to the methods of the invention. Alternatively, improvement in the disease may be classified as a partial response. By "partial response" is meant a reduction in all measurable tumor burden (i.e., the number of malignant cells present in a subject, or the measured tumor mass volume or number of abnormal monoclonal proteins) in the absence of new lesions of at least about 50%, which may last for 4-8 weeks, or 6-8 weeks.

Treatment according to the invention includes the use of a "therapeutically effective amount" of the drug. By "therapeutically effective amount" is meant an amount effective, at dosages and for durations of time required, to achieve the desired therapeutic effect.

The therapeutically effective amount may vary according to a number of factors, such as the disease state, age, sex, and weight of the individual, and the ability of the drug to exert a desired response in the individual. A therapeutically effective amount is also an amount that exceeds any toxic or detrimental effect of the antibody or antibody portion for which a therapeutically beneficial effect is sought.

A "therapeutically effective amount" for tumor therapy can also be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer can be assessed in an animal model system that predicts efficacy in human tumors.

Alternatively, this property of the composition can be assessed by an in vitro assay known to the skilled person which tests the ability of the compound to inhibit cell growth or induce apoptosis. A therapeutically effective amount of the therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. One of ordinary skill in the art will be able to determine such amounts based on factors such as: the size of the subject, the severity of the subject's symptoms and the particular composition or route of administration selected.

The dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased, as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form to facilitate administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suitable as unitary dosages for the subjects being treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

The specification for the dosage unit form of the invention is written by and directly dependent on the following factors: (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds for treatment of sensitivity in an individual.

Effective dosages and dosing regimens for the multispecific antibodies used in the present invention depend on the disease or condition to be treated and may be determined by one skilled in the art.

An exemplary, non-limiting range of therapeutically effective amounts of multispecific antibodies used in the present invention is about 0.1-100mg/kg, such as about 0.1-50mg/kg, for example about 0.1-20mg/kg, such as about 0.1-10mg/kg, for example about 0.5, such as about 0.3, about 1, or about 3 mg/kg. In another embodiment, the antibody is administered at a dose of more than 1mg/kg, such as a dose of 1-20mg/kg, e.g., a dose of 5-20mg/kg, e.g., a dose of 8 mg/kg.

The effective amount of the pharmaceutical composition required can be readily determined and prescribed by a medical professional having ordinary skill in the art. For example, a clinician or veterinarian can start a dose of drug employed in a pharmaceutical composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

In one embodiment, the multispecific antibody is administered by infusion at a weekly dose of 10-500mg/kg, such as 200-400 mg/kg. Such administration may be repeated, for example, 1 to 8 times, such as 3 to 5 times. Administration may be by continuous infusion over a period of 2-24 hours, such as 2-12 hours.

In one embodiment, if it is desired to reduce side effects, including toxicity, the multispecific antibody is administered by slow continuous infusion over a long period, such as over 24 hours.

In one embodiment, the multispecific antibody is administered up to 8 times, such as 4-6 times, at a weekly dose of 250mg-2000mg, such as, for example, 300mg,500mg,700mg,1000mg,1500mg or 2000 mg. The administration may be by continuous infusion over a period of 2-24 hours, such as 2-12 hours. Such a regimen may be repeated as often as desired, for example, after 6 months or 12 months. The dosage can be determined or adjusted after administration by, for example, taking a biological sample and measuring the amount of the compound of the invention in the blood using an anti-idiotype antibody targeting the antigen binding region of the multispecific antibody.

In a further embodiment, the multispecific antibody is administered once a week for 2-12 weeks, such as 3-10 weeks, such as 4-8 weeks.

In one embodiment, the multispecific antibody is administered by maintenance therapy, such as, for example, once a week for a period of 6 months or more.

In one embodiment, the multispecific antibody is administered by a regimen comprising one infusion of the multispecific antibody followed by an infusion of the multispecific antibody conjugated to a radioisotope. This protocol may be repeated, for example, after 7-9 days.

As non-limiting examples, the treatment according to the invention may be provided as a daily dose of the antibody in an amount of about 0.1-100mg/kg per day, such as at least one of 0.5,0.9,1.0,1.1,1.5,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,40,45,50,60,70,80,90 or 100mg/kg, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39 or 40 days, optionally 1,2,3, 7,8,9,10, 20, 12,13,14,15,16,17,18,19,20, 1, 21,22,23,24,25,26,27,28,29,30,31,32,33,34,35, 39 or 40 days, at least one of 12,13,14,15,16,17,18,19, or 20 weeks, or any combination thereof, a single or divided dose once every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof is used.

In some embodiments, the multispecific antibody molecule is used in combination with one or more other therapeutic agents, such as a chemotherapeutic agent. Non-limiting examples of DNA damaging chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin and analogs or metabolites thereof, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozotocin, dacarbazine, methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside analogs (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, guanine, pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include: paclitaxel, docetaxel and related analogs; vincristine, vinblastine, and related analogs; thalidomide, lenalidomide, and related analogs (e.g., CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF- κ B inhibitors including I κ B kinase inhibitors; antibodies that bind to proteins that are overexpressed in cancer and thus down-regulate cell replication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab; and other inhibitors of proteins or enzymes known to be up-regulated, overexpressed, or activated in cancer (their inhibition down-regulates cell replication).

In some embodiments, the antibodies of the invention may be utilized

(bortezomib) is administered prior to, concurrently with or after treatment.

All cited documents are specifically incorporated by reference herein in their entirety.

Although specific embodiments of the invention have been described above for purposes of illustration, it will be understood by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims.

Examples

The following examples are provided to illustrate the invention. These embodiments are not intended to limit the present invention to any particular application or principle of operation. For all constant region positions discussed in this invention, the numbering convention is according to the EU index as in Kabat (Kabat et al, 1991, Sequences of Proteins of Immunological Interest,5th ed., United States Public Health Service, National Institutes of Health, Bethesda, incorporated by reference in its entirety). Those skilled in the antibody art will appreciate that this convention consisting of non-sequential numbering in specific regions of immunoglobulin sequences can be used to refer to conserved positions in immunoglobulin families with standardization. Thus, the position of any given immunoglobulin as defined by the EU index does not necessarily correspond to its sequential sequence.

Example 1 design of non-native Charge substitutions to reduce pI

Antibody constant chains were modified with lower pI by engineering substitutions in the constant domains. The reduced pI can be engineered by replacing a basic amino acid (K or R) with an acidic amino acid (D or E), which results in the greatest reduction in pI. Mutations from basic to neutral amino acids and from neutral to acidic amino acids will also result in a decrease in pI. A list of amino acid pK values can be found in Table 1 of Bjellqvist et al, 1994, Electrophoresis 15: 529-539.

We chose to develop substitutions in the antibody CH1(C γ 1) and C L (Ckappa or CK) regions (sequences shown in figure 13) because, unlike Fc regions, they did not interact with natural ligands that affect the pharmacological properties of the antibody.

For all heterodimeric proteins herein, genes encoding the heavy and light chains of the antibody were constructed in mammalian expression vector pTT5 human IgG1 constant chain gene WAs obtained from IMAGE cloning and subcloned into pTT5 vector VH and V L genes encoding anti-VEGF antibodies were synthesized commercially (Blue Heron Biotechnologies, Bothell WA) and subcloned into vectors encoding the appropriate C L and IgG1 constant chains

Directed mutagenesis (Stratagene, L aJolla CA) used directed mutagenesis to construct amino acid modifications all DNA was sequenced to confirm the fidelity of the sequence.

Plasmids containing the heavy chain gene (VH-C γ 1-C γ 2-C γ 3) and the light chain gene (V L-C κ) were co-transfected into 293E cells using cationic liposomes (lipofectamine) (Invitrogen, Carlsbad CA) and the cells were grown in FreeStyle 293 medium (Invitrogen, Carlsbad CA). after 5 days of growth, antibodies were affinity purified from the culture supernatants by protein A using MabSelect resin (GE Healthcare).

pI engineered mabs are typically characterized by SDS PAGE on an Agilent Bioanalyzer, by Size Exclusion Chromatography (SEC), isoelectric focusing (IEF) gel electrophoresis, by Biacore binding to antigen, and Differential Scanning Calorimetry (DSC). All mAbs showed high purity on SDS-PAGE and SEC. IEF gels indicate that each variant has a designed isoelectric point. Binding analysis on a large Biacore showed that the pI engineered variants bound antigen with similar affinity to the parent antibody, indicating that the designed substitutions did not interfere with mAb function. DSC in the figure shows which variants have substantially high thermostability.

Depending on the circumstances in which pharmacokinetic experiments of serum half-life were performed in B6 mice, B6 mice were homozygous for murine FcRn and heterozygous for human FcRn (mFcRn-/-, hFcRn +) (Petkova et al, 2006, Int Immunol18(12):1759-69, incorporated by reference in its entirety), referred to herein as hFcRn or hFcRn + mice.

Groups of 4-7 female mice randomized by weight (20-30g range) were given a single intravenous tail vein injection of antibody (2mg/kg), blood was aspirated from the orbital plexus at each time point (-50 ul), processed into serum, and stored at-80 ℃ until analysis, antibody concentrations were determined using the E L ISA assay, antibody serum concentrations were measured using recombinant antigen as the capture reagent, and detected using biotinylated anti-human kappa antibody and europium labeled streptavidin, time resolved fluorescence signals were collected, individual mice were assayed using the non-compartmental model using WinNon L in (Pharsight Inc, Mountain View CA), PK parameters were used together with uniform weighting of the points.

Example 2 modification of the pI of the constant region

Reduction of pI of a protein or antibody can be performed using a variety of means. At the highest basic levels, residues with high pKa (lysine, arginine and to some extent histidine) are replaced by neutral or negatively charged residues, and/or neutral residues are replaced by low pKa residues (aspartic acid and glutamic acid). The particular substitution may depend on many factors, including location in structure, position in function, and immunogenicity.

Because immunogenicity is an issue, some efforts may be made to minimize the risk that substitutions that reduce pI will cause immunogenicity. One way to reduce the risk is to minimize the mutation load of the variant, i.e. to reduce the pI with a minimum number of mutations. Charge exchange mutations, in which K, R, or H is replaced by D or E, have the greatest effect on reducing pI, so these substitutions are preferred. Another way to minimize the risk of immunogenicity while reducing pI is to use substitutions from homologous human proteins. Thus isotype differences between IgG subclasses (IgG1, IgG2, IgG3, and IgG4) provide low risk substitutions for antibody constant chains. Since immune recognition occurs at the local sequence level, i.e., MHC II and T-cell receptors recognize epitopes typically 9 residues in length, pI-altered substitutions can be accompanied by isotype substitutions proximal in sequence. In this way, the epitope can be extended to match the native isoform. Such substitutions would therefore constitute epitopes present in other human IgG isotypes and would therefore be expected to be tolerated.

One way to engineer pI changes is to use isotype switching, as described herein.

Another way to engineer lower pI into proteins and antibodies is to fuse negatively charged residues to the N-or C-terminus. Thus, for example, a peptide consisting essentially of aspartic acid and glutamic acid can be fused to the N-terminus or C-terminus of the antibody heavy chain, light chain, or both. The C-terminus is preferred because the N-terminus is structurally close to the antigen binding site.

Based on the manner of engineering, many variants are designed to alter the isoelectric point of the antibody heavy chain (typically the Fc region) and in some cases the light chain.

Example 3 isotype light chain constant region variants

The homology between CK and C λ is not as high as between IgG subclasses, however the existing sequence and structural homology is still used to direct substitutions to form an isotype low-pI light chain constant region. In fig. 56, the positions of residues with contributing to higher pI (K, R and H) or lower pI (D and E) are highlighted in bold. Grey indicates lysine, arginine and histidine, preferably with aspartic acid or glutamic acid, which can be substituted to lower the isoelectric point. These variants, alone or in combination, can be independently and optionally combined with all other heavy chain variants in the framework with at least one light chain.

Example 4. purification mixture of antibody variants with altered isoelectric points.

Substitutions that alter the isoelectric point of the antibody can be introduced into one or more chains of the antibody variant to facilitate analysis and purification. For example, heterodimeric antibodies such as those disclosed in US2011/0054151a1 may be purified by varying the isoelectric point of one chain, such that multiple species present after expression and protein a purification may be purified by methods that separate proteins based on charge differences (such as ion exchange chromatography).

As an example, the heavy chain of bevacizumab is altered by introducing a substitution to lower its isoelectric point so that the difference in charge between the three species produced when WT-IgG1-HC, low pI-HC and WT-L C are transfected in 293E cells is large enough to facilitate purification by anion exchange chromatography as described above, and the transfection and primary purification by protein a chromatography are also described above the sequences of three species "heavy chain 1 of XENP 10653", "heavy chain 2 of XENP 10653", and "light chain of XENP 10653" are in the figures after protein a purification, three species with almost the same molecular weight but different charge are obtained, these are WT-IgG1-HC/WT-IgG1-HC homodimer (pI ═ 8.12), WT-1-HC/l low-pI-HC heterodimer (pI ═ 6.89) and low-pI-HC/l-HC-pI-HC heterodimer (pI-HC-8.89) which are in a low-pH range of WT-hci species (pI-HC/l) and which are not in a low-pH-C/il-HC-C-b (b-b (b) and b (b) when b) are in a C (b) when b) are in the same as in.

Example 5 design of non-native Charge substitutions to alter pI

The pI of the antibody constant chain was altered by engineering substitutions in the constant domain. The reduced pI can be engineered by replacing a basic amino acid (K or R) with an acidic amino acid (D or E), which results in the greatest reduction in pI. Mutations from basic to neutral amino acids and from neutral to acidic amino acids will also result in a decrease in pI. Conversely, an increased pI can be engineered by replacing an acidic amino acid (D or E) with a basic amino acid (K or R), which results in the greatest increase in pI. Mutations from acidic to neutral amino acids and from neutral to basic amino acids will also result in an increase in pI. A list of amino acid pK values can be found in Table 1 of Bjellqvist et al, 1994, Electrophoresis 15: 529-539.

In deciding which position to mutate, the surrounding environment and the number of contact points of the WT amino acid with its neighbors are taken into account, such as to minimize the effect of one or a set of substitutions on structure and/or function. The fraction of solvent accessibility or exposure at each constant region location is calculated using the relevant crystal structure. Based on such analysis, many substitutions were identified that reduce or increase the pI but are predicted to have minimal impact on the biophysical properties of the domain.

The calculation of the pI of the protein was performed as follows. First, the number of D, E, C, H, K, R and Y amino acids and the number of N-and C-termini present in the protein were counted. The pI was then calculated by identifying the pH at which the protein had a total charge of 0. This is done by calculating the net charge of the protein at a number of pH values tested. The test pH was set in an iterative manner, increasing from a low pH of 0 to a high pH of 14 in 0.001 increments until the charge of the protein reached or exceeded 0. The net charge of the protein at a given pH is calculated by the following equation:

wherein q is_Protein(pH) is the net charge on the protein at a given pH, is the number of amino acids i (or N-or C-termini) present on the protein, and is the pK of amino acid i (or N-or C-termini).

Example 6 purification mixtures of antibody variants with altered isoelectric points

The variants were first purified by protein A and then loaded onto GE healthcare HiTrap SP HP cation exchange columns in 50mM MES (pH 6.0) and eluted with a NaCl gradient after elution fractions from each peak were loaded onto L onza Isogel IEF plates (pH range 7-11) for analysis.

Example 7 stability of pI isostere variants

DSF experiments were performed using a Bio-Rad CFX Connect real-time PCR detection system protein was mixed with SYPRO orange fluorochrome and diluted to 0.25 or 0.50mg/m L in PBS the final concentration of SYPRO orange was 10X after an initial 10 minute incubation period at 25 ℃, protein was heated from 25 to 95 ℃ using a heating rate of 1 ℃/min, fluorescence measurements were taken every 30 seconds.

Example 8 design of charged scFv linkers capable of IEX purification of heterodimeric bispecific antibodies containing scFv

For heterodimeric bispecific antibodies containing scfvs (examples are shown in the figures), the other region where charged substitutions are introduced is the scFv linker linking VH and V L of the scFv construct the most common linker used is (GGGGS)3 or (GGGGS)4, which has shown sufficient flexibility to allow stable formation of scFv without diabody formation, these sequences are often non-natural and contain little sequence specificity for possible immunogenic epitopes.

The biophysical behavior of the charged linker in the scFv-His format was first assessed and then later constructed in the anti-CD 19xCD3 Fab-scFv-Fc bispecific format. Genes encoding scFv of anti-CD 3 antibody SP34 or an engineered form of anti-CD 194G 7 antibody were constructed in mammalian expression vector pTT 5. For the full-length construct, the human IgG1 constant chain gene was obtained from IMAGE clonopathy and cloned into the pt 5 vector. The scFv gene WAs synthesized commercially (Blue Heron Biotechnologies, Bothell WA). Use of

Directed mutagenesis (Stratagene, L aJolla CA) used directed mutagenesis to construct amino acid modifications all DNA was sequenced to confirm the fidelity of the sequence.

Plasmids containing scFv or heavy and light chain genes were transfected (or co-transfected for the full-length version) into 293E cells using cationic liposomes (Invitrogen, Carlsbad CA) and cells were grown in FreeStyle 293 medium (Invitrogen, Carlsbad CA). After 5 days of growth, the antibody was purified from the culture supernatant by protein a (full length) using MabSelect resin (GEHealthcare) or Ni-NTA resin for His-tagged scFv. Further purified by ion exchange chromatography (IEX) to assess the ability of the altered pI heavy chain to achieve efficient purification. An example of IEX purification for an anti-CD 19xCD3 bispecific antibody containing a positively charged linker in the CD3 scFv is shown in the figure. Antibody concentrations were determined by the bicinchoninic acid (BCA) assay (Pierce).

The pI engineered scFv or antibody is characterized by SDS PAGE, Size Exclusion Chromatography (SEC), isoelectric focusing (IEF) gel electrophoresis, and/or Differential Scanning Fluorescence (DSF).

Example 9 stability and behavior of scFv containing charged linker

The SEC behavior of anti-CD 3 scFv and anti-CD 19 scFv containing positively or negatively charged linkers, respectively, was evaluated and stability was evaluated using DSF.differential Scanning Fluorescence (DSF) was used to evaluate the stability of scFv containing a charged linker.DSF experiments were performed using a Bio-Rad CFX Connect real-time PCR detection system.proteins were mixed with SYPRO orange fluorescent dye and diluted to 0.25 or 0.50mg/m L in PBS.SYPRO orange at a final concentration of 10 X.after an initial 10 minute incubation period at 25 ℃, the protein was heated from 25 to 95 ℃ using a heating rate of 1 ℃/min.

The positively charged scFv linker on the anti-CD 3 scFv in the anti-CD 19xCD3 Fab-scFv-Fc construct had the unexpected property of reducing the amount of high molecular weight aggregation (figure). SEC chromatograms of two bispecific constructs incubated at various concentrations (13121-with a standard (GGGGS)4 linker) and (13124-with a charged linker (GKPGS)4) confirm this.

The activity of the anti-CD 19xCD3 construct containing a charged scFv linker in the anti-CD 3 scFv was evaluated using the RTCC assay with PBMC and a Fab-scFv-Fc format bispecific anti-CD 19xCD3 antibody containing a different scFv linker (FIG.). The linker had little effect on RTCC activity, with the exception of highly charged linker (gkgkgkks) 3, which had lower activity.

The sequences of all constructs of the invention are shown in the figure.

Claims (15)

1. A heterodimeric antibody comprising:

a) a first monomer comprising:

i) a first variant human IgG heavy chain constant domain comprising a first variant Fc domain; and

ii) a single chain Fv region (scFv) comprising a first heavy chain variable domain and a first light chain variable domain linked together by an scFv linker; and

b) a second monomer comprising:

i) a second heavy chain variable domain; and

ii) a second variant human IgG heavy chain constant domain comprising a second variant Fc domain; and

c) a light chain comprising a second light chain variable domain,

wherein the first variant Fc domain has only the amino acid substitutions E233P, L234V, L235A, G236del, S364K, S267K and E357Q,

wherein the second heavy chain variable domain and the second light chain variable domain form an antigen binding domain,

wherein the second variant human IgG heavy chain constant domain has only the amino acid substitutions N208D, E233P, L234V, L235A, G236del, S267K, Q295E, L368D, K370S, N384D, Q418E and N421D, and

wherein the numbering is according to the EU index as Kabat.

2. A heterodimeric antibody comprising:

a) a first monomer comprising:

i) a first variant human IgG heavy chain constant domain comprising a first variant Fc domain; and

ii) a single chain Fv region (scFv) comprising a first heavy chain variable domain and a first light chain variable domain linked together by an scFv linker; and

b) a second monomer comprising:

i) a second heavy chain variable domain; and

ii) a second variant human IgG heavy chain constant domain comprising a second variant Fc domain; and

c) a light chain comprising a second light chain variable domain,

wherein the first variant Fc domain has only the amino acid substitutions E233P, L234V, L235 38235 235A, G236del, S364K, S267K, E357Q, M428L and N434S,

wherein the second heavy chain variable domain and the second light chain variable domain form an antigen binding domain,

wherein the second variant human IgG heavy chain constant domain has only the amino acid substitutions N208D, E233P, L234V, L235A, G236del, S267K, Q295E, L368D, K370S, N384D, Q418E, N421D, M428L, and N434S, and

wherein the numbering is according to the EU index as Kabat.

3. A heterodimeric antibody according to claim 1 or 2 wherein said scFv comprises a charged scFv linker.

4. A heterodimeric antibody according to claim 3 wherein said scFv linker is GKPGSGKPGSGKPGSGKPGS.

5. A heterodimeric antibody according to claim 1 or 2 wherein said scFv binds CD 3.

6. The heterodimeric antibody of claim 5, wherein the scFv comprises a variable heavy chain domain (vh domain) and a variable light chain domain (vl domain), and wherein the vh domains comprise vhCDR1, vhCDR2, and vhCDR3, wherein the vhCDR1 consists of SEQ ID NO:411, the vhCDR2 consists of SEQ ID NO:413, and the vhCDR3 consists of SEQ ID NO: 417; the vl domain comprises vlCDR1, vlCDR2 and vlCDR3, wherein said vlCDR1 consists of SEQ ID NO:420, said vlCDR2 consists of SEQ ID NO:425, and said vlCDR3 consists of SEQ ID NO: 433.

7. A heterodimeric antibody according to claim 6 wherein the variable heavy chain (vh) domain of said scFv consists of SEQ ID NO:397 and the variable light chain (vl) domain of said scFv consists of SEQ ID NO: 398.

8. A heterodimeric antibody according to claim 6 wherein said scFv consists of SEQ ID NO 643.

9. A nucleic acid composition comprising:

a) a first nucleic acid encoding a first monomer according to claim 1 or 2;

b) a second nucleic acid encoding a second monomer according to claim 1 or 2; and

c) a third nucleic acid encoding the light chain of claim 1 or 2.

10. The nucleic acid composition of claim 9, comprising

a) A first expression vector comprising the first nucleic acid;

b) a second expression vector comprising the second nucleic acid; and

c) a third expression vector comprising said third nucleic acid.

11. A host cell comprising the nucleic acid composition of claim 9.

12. A host cell comprising the nucleic acid composition of claim 10.

13. A method of making a composition, the method comprising culturing the host cell of claim 11 under conditions in which the nucleic acid is expressed and recovering the composition.

14. A method of making a composition, the method comprising culturing the host cell of claim 12 under conditions in which the nucleic acid is expressed and recovering the composition.

15. A heterodimeric antibody according to claim 1 or 2 for use in therapy.

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